Compounds, processes for the preparation thereof and anti-tumor agents

ABSTRACT

This invention relates to pyridone-carboxylic acid derivatives of the following formula or salts thereof: ##STR1## wherein R 1  is a hydrogen atom, a halogen atom, etc., 
     R 2  is a carboxyl group etc., 
     R 3  is a hydrogen atom etc., 
     A is a nitrogen atom or CH, 
     m is 1 or 2, and 
     Y is an eliminable group or a group having the following formula: ##STR2## wherein R 4  is a hydrogen atom or a lower alkyl group, 
     Z is a hydrogen atom, a lower alkyl group, etc., 
     R 5  is a hydrogen atom, a lower alkyl group, etc., 
     n is 0 or 1, and 
     p is 1, 2, 3 or 4 
     and to processes for the preparation of these compounds, and further to anti-tumor agents which contain the above compounds as effective ingredients.

This application claims priority under 35 U.S.C. § 371 fromPCT/JP95/01110, filed Jun. 6, 1995.

TECHNICAL FIELD

This invention relates to novel pyridone-carboxylic acid derivatives,anti-tumor agents containing the same as effective ingredients, andprocesses for the preparation of the novel pyridone-carboxylic acidderivatives, etc.

BACKGROUND ART

There have been known both various pyridone-carboxylic acid derivativesper se which have 2-thiazolyl groups and the fact that thesepyridone-carboxylic acid derivatives show anti-bacterial activity. Forexample, the following Compound A is disclosed in Example 24 of JapanesePatent Application Laid-Open (Kokai) No. 152682/1986 (hereinafterreferred to as Ref. 1): ##STR3##

In Example 5 of said Ref. 1, the following Compound B is exhibited:##STR4##

Further, in Example 12 of Ref. 1, the following Compound C is disclosed:##STR5##

The above compounds B and C are also disclosed in Table 1 of JapanesePatent Application Laid-Open (Kokai) No. 33176/1987 (hereinafterreferred to as Ref. 2).

Moreover, the following Compound D is mentioned in Example 24-4 ofJapanese Patent Application Laid-Open (Kokai) No. 56959/1985(corresponding to European Patent Application Laid-Open No. 131839 andU.S. Pat. No. 4,730,000; hereinafter referred to as Ref. 3 together) andin Example 15 of Japanese Patent Application Laid-Open (Kokai) No.251667/1986 (hereinafter referred to as Ref. 4). ##STR6##

The following Compound E is mentioned in Example 28-16 of JapanesePatent Application Laid-Open (Kokai) No. 163866/1985 (corresponding toEuropean Patent Application Laid-Open No. 154780 and U.S. Pat. No.4,774,246; hereinafter referred to as Ref. 5 together). ##STR7##

Further, the following Compound F is shown in Example 8 of JapanesePatent Application Laid-Open (Kokai) No. 85255/1990 (hereinafterreferred to as Ref. 6). ##STR8##

However, the chemical structure of these compounds is different from thestructure of Compound (I) of this invention in the following points (1)and (2).

(1) The 6-position of Compounds A to F is always substituted with afluorine atom.

(2) The substituent at the 7-position of Compounds A, C, D, E and F isnot a substituted 1-pyrrolidinyl group.

In addition, Refs. 1 to 6 only mention that these Compounds A to Fexhibit antibacterial activity, and teach nothing on their anti-tumoractivity or anti-cancer activity.

It is known that certain kinds of pyridone-carboxylic acid derivativesshow anti-tumor activity or anti-cancer activity. For example, CancerResearch 52, 2818 (1992) reports that the following Compound G hasanti-tumor activity: ##STR9##

It is reported in this treatise that 90 kinds of pyridone-carboxylicacid derivatives were investigated for anti-tumor activity, and thatmost of said derivatives showed no anti-tumor activity with theexception of only several types of the derivatives. It is further taughtthat the cyclopropyl group which is substituted at the 1-position andthe two halogen atoms which are each substituted at the 6- and8-positions play an important role for the expression of anti-tumoractivity, and that a pyridone-carboxylic acid derivative which is freefrom such substituents shows no anti-tumor activity.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 to 9 show the change of tumor growth inhibition rate (IR) withthe lapse of days in the case where a compound of this invention wasadministered to nude mice into which various human cancer cells had beentransplanted.

FIGS. 10 to 14 show the change of tumor weight with the lapse of days inthe above experiment.

Such numerals in each Figure as "1--1 (50) 91!" each mean in this order"Compound Number, (Administered Amount mg/kg), Tumor Growth InhibitionRate IR % on the last day of observation!.

FIGS. 15 to 19 show reaction formulae of the reactions which arementioned in Example of Series A Production of Intermediates of thisInvention!, Example of Series B Production of Raw Materials of thisInvention! and Example of Series C Production of Compounds of thisInvention! which will be mentioned later.

DISCLOSURE OF INVENTION

Seeking intensively for compounds having anti-tumor activity, thepresent inventors have resultantly found that novel pyridone-carboxylicacid derivatives having such 2-thiazolyl groups as may be substitutedshow remarkable anti-tumor activity.

This invention relates to pyridone-carboxylic acid derivatives whichhave 2-thiazolyl groups and which have the following general formula(I). and to salts thereof: ##STR10## wherein R₁ is a hydrogen atom, alower alkoxy group, a halogen atom, a lower alkyl group which may besubstituted with halogen atom, or a phenyl group which may besubstituted with halogen atom;

R₂ is a carboxyl group or a group convertible to a carboxyl group;

R₃ is a hydrogen atom, an amino group which may be protected, a halogenatom or a lower alkyl group which may be substituted with halogen atom;

A is nitrogen atom or CH;

m is an integer of 1 or 2; and

Y is an eliminable group or a group having the following formula Y'##STR11## wherein R₄ is a hydrogen atom or a lower alkyl group;

Z is a hydrogen atom, a lower alkyl group or a group convertible to ahydrogen atom;

R₅ is a hydrogen atom, a halogen atom, a lower alkoxy group, a loweralkylthio group or a lower alkyl group which may be substituted withhalogen atom;

n is an integer of 0 or 1; and

p is an integer of 1, 2, 3 or 4.

The compounds of this invention include, of course, their stereoisomers,optical isomers, hydrates, solvates, etc.

The compounds (I) of this invention are classified into two categoriesaccording to the character of the substituents.

One of the categories includes compounds of formula (I) wherein Y is an"eliminable group", and these compounds are useful as directintermediates for compounds wherein Y is the above Y'. Thus, one of theobjects of this invention lies in providing intermediates forpyridone-carboxylic acid derivatives which are useful as anti-tumoragents.

As the "eliminable group" included in the definition of substituent Y,any group can be used so long as it can be substituted with thelater-described pyrrolidine derivative (III) and thereby eliminated, andexamples of such a group include a halogen atom, a lower alkoxy group, alower alkylthio group, a lower alkylsulfinyl group, a loweralkylsulfonyl group, an arylsulfonyl group, a lower alkylsulfonyloxygroup, an arylsulfonyloxy group, etc. Among these groups, halogen atomssuch as fluorine atom and chlorine atom are preferred. As for othersubstituents or salts, concrete examples thereof will be seen in theexplanation of Compound (I-a) of this invention below.

The compounds of this invention in another category are compounds of theabove formula (I) wherein substituent Y is Y', and they are useful asexcellent anti-tumor agents or anti-cancer agents. Thus, this inventionprovides pyridone-carboxylic acid derivatives having the followingformula (I-a), physiologically acceptable salts thereof, methods toproduce such derivatives and salts, and anti-tumor agents which containsaid derivatives or salts as effective ingredients: ##STR12## wherein A,R₁, R₂, R₃, R₄, R₅, Z, m, n and p are as defined above.

Salts of the compounds having formula (I-a) include both salts derivedfrom the carboxyl group portion which is contained in the definition ofR₂ of formula (I-a) and acid addition salts derived from the basicsubstituent group portion which is bound to the 3-position of the1-pyrrolidinyl group.

Examples of said salts at the carboxyl group portion include salts withmetals such as sodium, potassium, magnesium, zinc, silver, aluminum andplatinum, and salts with organic bases such as dimethyl-aminoethanol,methylaminoethanol, triethanolamine and guanidine.

As for examples of acid addition salts at the basic substituent groupwhich is bound to the 3-position of the 1-pyrrolidinyl group of formula(I-a), there can be taken salts with inorganic acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid andphosphoric acid, and salts with organic acids such as oxalic acid,maleic acid, fumaric acid, malonic acid, lactic acid, malic acid, citricacid, tartaric acid, benzoic acid, methanesulfonic acid,p-toluenesulfonic acid, ascorbic acid, glucuronic acid,2-hydroxy-ethanesulfonic acid, lactobionic acid and glucoheptonic acid.

Each of the above substituents is explained in the following:

In this specification, "lower alkyl group" means a straight-chain alkylgroup or branched one having 1 to 5 carbon atoms, and examples of such agroup include methyl group, ethyl group, n-propyl group, isopropylgroup, n-butyl group, t-butyl group, etc. "Lower alkoxy group" meansalkoxy group having 1 to 5 carbon atoms, and preferable examples aremethoxy group and ethoxy group.

Examples of "halogen atom" include chlorine atom, fluorine atom andbromine atom.

Substituent R₁ in formula (I-a) is located at the 4- and/or 5-positionof 2-thiazolyl group, and preferable examples include hydrogen atom,halogen atom such as fluorine atom, chlorine atom and bromine atom,lower alkoxy group such as methoxy group and ethoxy group, lower alkylgroup such as methyl group and ethyl group, lower alkyl groupsubstituted with halogen atom such as trifluoromethyl group, and phenylgroup which may be substituted with halogen atom such as3,4-difluorophenyl group.

As the "group convertible to a carboxyl group" contained in thedefinition of substituent R₂, any group can be used so long as it isconvertible to a carboxyl group by a chemical means or an enzymologicalmeans, and preferable examples of such a group include hydroxy-methylgroup, formyl group, ester form and physiologically acceptable salts ofcarboxyl group.

Examples of ester forms which are convertible to carboxyl groups mainlyby a chemical means include lower alkyl esters such as methyl ester andethyl ester.

Examples of ester forms which are convertible to carboxyl group not onlythrough a chemical means but through an enzymological means includelower alkanoyloxy lower alkyl esters such as acetoxymethyl ester,1-acetoxyethyl ester and pivaloyloxymethyl ester; loweralkoxycarbonyloxy lower alkyl esters such as 1-ethoxycarbonyloxyethylester; di-lower alkylamino lower alkyl esters such as2-dimethylaminoethyl ester; 2-(1-piperidinyl)ethyl ester;3-butyrolactonyl ester; choline ester; phthalidyl ester;(5-methyl-2-oxo-1,3-dioxol-4-yl) methyl ester, etc.

Preferable examples of substituent R₃ include hydrogen atom, halogenatom such as fluorine atom and chlorine atom, amino group, amino groupprotected by amino-protecting group, and lower alkyl group substitutedwith halogen atom such as trifluoromethyl group. As saidamino-protecting group, any protecting group can be employed so long asit is easily eliminated by a usual deprotecting reaction such ashydrolysis or hydrogenolysis without giving any substantial influence onother structural portions. Concretely, said protecting group issubstantially the same as the "group convertible to hydrogen atom" inthe definition of substituent Z which is explained later. Preferableexamples of amino-protecting group include benzyl group and tritylgroup.

Substituent R₄ is hydrogen atom or lower alkyl group, and preferablyemployed are hydrogen atom, methyl group and ethyl group. Incidentally,the 3-position of the 1-pyrrolidinyl group to which the basicsubstituent group possessing R₄ and Z is bound is occupied by anasymmetric carbon atom, which can cause optical isomers to be existent.

Preferable examples of substituent Z include hydrogen atom, lower alkylgroup such as methyl group and ethyl group, or "group convertible to ahydrogen atom". As said "group convertible to a hydrogen atom", anygroup can be used so long as it is convertible to a hydrogen atom by achemical means such as hydrolysis and hydrogenolysis or by an enzymological means.

Examples of Z as such a "group convertible to a hydrogen atom" include,first, hydrolyzable groups. Concrete examples of hydrolyzable groupsinclude acyl groups, groups having an oxycarbonyl group, amino acidresidues and peptide residues, and further, for example,o-nitrophenylsulfenyl, trimethylsilyl, tetrahydro-pyranyl,diphenylphosphinyl, etc. In general, Compounds (I-a) of this inventionwherein Z is an amino acid residue or peptide residue as a groupconvertible to a hydrogen atom are superior, in solubility, to thosewherein Z is neither amino acid residue nor peptide residue, and areadvantageously used in the form of liquid agents such as an injection.

Examples of the above-mentioned acyl groups include formyl, acetyl,trifluoroacetyl, etc.

Further, examples of the aforementioned groups having an oxycarbonylgroup include ethoxycarbonyl, t-butoxycarbonyl (CH₃)₃ C--OCO--!,benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, vinyloxycarbonyl,β-(p-toluenesulfonyl)ethoxycarbonyl, etc.

Further, examples of amino acid residues include amino acid residues perse and such amino acid residues as are protected with a protecting groupwhich is usually employed in peptide synthesis. Examples of protectinggroups for an amino group usually employed in peptide synthesis includeacyl groups such as formyl and acetyl, arylmethyloxycarbonyl groups suchas benzyloxycarbonyl and p-nitrobenzyloxycarbonyl, a t-butoxycarbonylgroup (CH₃)₃ C--OCO--!, etc.

As for amino acid residues, there can be used any, e.g., an alanineresidue CH₃ CH(NH₂)CO--! and a leucine residue (CH₃)₂ CHCH₂CH(NH₂)CO--!. In general, these amino acids are represented by a set ofthree English letters, and this principle is followed also in thepresent specification. Further, L-forms, D-forms or their mixtures aredistinguished by the addition of symbol "L-", "D-" or "DL-" on the headof the three letters. These symbols are omitted when these isomers arereferred to as a whole.

Concrete examples of amino acid residues include residues of such aminoacids as Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine),Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His(histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Met(methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr(threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva(norvaline), Hse (homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl(5-hydroxylysine), Orn (ornithine) and β-Ala.

Two to five, preferably two to three of the aforesaid amino acids formthe peptide residues. As examples of such peptide residues, there can betaken residues of such peptides as Ala-Ala CH₃CH(NH₂)CO--NHCH(CH₃)CO--!, Gly-Phe, Nva-Nva, Ala-Phe, Gly-Gly,Gly-Gly-Gly, Ala-Met, Met-Met, Leu-Met and Ala-Leu.

The residues of these amino acids or peptides can take stereochemicalconfiguration of D-form, L-form or a mixture thereof, but L-form ispreferred. Further, when Z is a residue of an amino acid or peptide,also such a residue may have an asymmetric carbon atom undercircumstances. Examples of amino acid residues having an asymmetriccarbon atom include residues of such amino acids as Ala, Leu, Phe, Trp,Nva, Val, Met, Ser, Lys, Thr and Tyr, and, as examples of peptideresidues which have an asymmetric carbon atom, there can be taken suchones as have, as a constituent ingredient, these amino acid residueshaving an asymmetric carbon atom.

Further, the group Z "convertible to a hydrogen atom" can be areductively eliminable hydrogenolyzable group, and examples of such agroup include arylsul fonyl groups such as o-toluenesul fonyl; methylgroups substituted with phenyl or benzyloxy such as benzyl, trityl andbenzyloxymethyl; arylmethoxycarbonyl groups such as benzyloxycarbonyland o-methoxy-benzyloxycarbonyl; and halogenoethoxycarbonyl groups suchas β, β, β-tri chloroethoxycarbonyl and β-iodoethoxycarbonyl, etc.

The substituent "(R₅)_(p) -" of 1-pyrrolidinyl group of formula Y' isbound to the pyrrolidine ring of said 1-pyrrolidinyl group. The mark pdenotes an integer of 1 to 4. When p is 2 to 4, R₅ may be identical ordifferent. R₅ may be bound to any position of the 1-pyrrolidinyl group,but preferably either to the position to which the basic substituentcontaining both R₄ and Z is bound (which position is hereinafter calledthe 3-position of 1-pyrrolidinyl group) or to the position adjacentthereto (hereinafter called the 2- and/or 4-position of 1-pyrrolidinylgroup). When R₅ is bound to a position other than the 3-position of the1-pyrrolidinyl group, and when R₅ is not a hydrogen atom, it followsthat said 1-pyrrolidinyl group have at least two asymmetric carbonatoms, with the result that the Compounds (I-a) of this invention canexist as a stereoisomer (cis form or transform) and an optical isomer.Preferable examples of substituent R₅ include hydrogen atom, lower alkylgroups such as methyl group and ethyl group, lower alkyl groupssubstituted with halogen atom such as fluoromethyl group andtrifluoromethyl group, lower alkoxy group such as methoxy group andethoxy group, lower alkylthio groups such as methylthio group, andhalogen atom such as chlorine atom and fluorine atom.

The compounds (I-a) of this invention having such substituents as havebeen detailedly described in the above and their physiologicallyacceptable salts are novel and excellent in anti-tumor activity.

In the compounds of this invention having the general formula (I-a),core compounds which show anti-tumor activity have the following formula(I-b) ##STR13## wherein R₁, m, R₄, n, R₅, p and A are as defined informula (I-a); R₃ ' is a hydrogen atom, amino group, halogen atom orlower alkyl group which may be substituted with halogen atom; and Z' isa hydrogen atom or lower alkyl group.

In the following, compounds represented by the above formula (I-b) willsometimes be abbreviated as "active compounds". When a compound which isnot an active compound is administered into a living body, said compoundis converted to an active compound in the living body undercircumstances. In such a case, the compound which is not an activecompound is sometimes abbreviated as a "prodrug". In this invention,examples of such a prodrug include compounds of formula (I-a) wherein Zis an amino acid residue or a peptide residue, or wherein R₂ is a formylgroup or an ester form such as acetoxymethoxycarbonyl.

Further, in the description of the present specification and claims,compounds which are convertible to active compounds by any means such asa chemical means or an enzymological means are generally referred to as"convertible compounds".

The structural characteristics of the compounds (I-a) of this inventionlie in that said compounds have the following constitution:

(1) the compounds have, as a basic skeleton, a pyridone carboxylic acidrepresented by the following formula 1 ##STR14## wherein A is as definedabove, and a, b, c and d denote positions at which substituents arebound,

(2) 2-thiazolyl group which may have a substituent is bound to theposition "a",

(3) a carboxyl group or a group convertible to a carboxyl group is boundto the position "b",

(4) the position "c" is unsubstituted, or a group such as amino group isbound to said position,

(5) A is either nitrogen atom or carbon atom which is not substitutedwith halogen atom such as fluorine atom, and

(6) the position "d" is substituted with a specific 1-pyrrolidinyl groupwhich has at least a substituent represented by the following formula 2##STR15## wherein R₄, Z and n are as defined above.

The compounds (I-a) of this invention are novel compounds which arestructurally characterized in particular by the combination of thesubstituents which are each bound to the positions "a" and "d" and bythe fact that A contains no fluorine atom.

All of the compounds of this invention included in formula (I-a) andtheir physiologically acceptable salts are excellent anti-tumor agentsor anti-cancer agents. In particular, compounds wherein A is CH arepreferable as anti-tumor agents. Much preferable are compounds wherein Ais CH, m and p are 1 and n is 0.

Especially preferable are compounds wherein A is CH, m and p are 1, n is0, R₁ is a hydrogen atom, R₂ is a carboxyl group, R₃ is a hydrogen atom,R₄ is a hydrogen atom or a lower alkyl group, Z is a hydrogen atom, andR₅ is a hydrogen atom, lower alkyl group or lower alkoxy group. Examplesof such compounds include1,4-dihydro-7-(3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid (see Compound 1-1 etc. in the later-described Table 1) andcompounds convertible thereto.

Although concrete examples of preferable compounds included in formula(I-a) are mentioned in Examples later, the following compounds andcompounds convertible thereto can be further taken as preferableexamples of 1,8-naphthyridine type compounds of formula (I-a) of thisinvention:

7-(3-amino-4-fluoro-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-4-methoxy-3-methyl-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-4-methoxy-4-methyl-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-3-fluoromethyl-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-4-fluoromethyl-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-4-trifluoromethyl-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-1-pyrrolidinyl)-1-(4-chloro-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-1-pyrrolidinyl)-1-(4,5-difluoro-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid

5-amino-7-(3-amino-1-pyrrolidinyl)-1-(4-fluoro-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-1-pyrrolidinyl)-1-4-dihydro-4-oxo-1-(4-trifluoromethyl-2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-1-pyrrolidinyl)-1-4-(3,4-difluorophenyl)-2-thiazolyl!-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid

7-(3-amino-1-pyrrolidinyl)-1-(5-bromo-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid

The following compounds and compounds convertible thereto can be takenas examples of pyridopyrimidine type compounds of formula (I-a) of thisinvention:

5,8-dihydro-2-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-5-oxo-8-(2-thiazolyl)pyrido2,3-d!-pyrimidine-6-carboxylic acid

8-(4-fluoro-2-thiazolyl)-5,8-dihydro-2-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-5-oxopyrido2,3-d!pyrimidine-6-carboxylic acid

PHARMACOLOGICAL TESTS TEST EXAMPLES

The anti-tumor activity of Compounds (I-a) of this invention isdescribed in the following. As controls, there were adopted bothCompound A which is disclosed in Japanese Patent Application Laid-openNo. 152682/1986 (Ref. 1) which is referred to in the beginning part ofthis specification, and Etoposide, i.e., a commercially availableanti-cancer agent, which has the later-described structural formula.

Test Example 1

In vitro anti-tumor activity (IC₅₀ :μg/ml) against murine P388lymphocytic leukemia cells

Test compounds were tested for anti-tumor activity according to MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide! method,using murine P388 lymphocytic leukemia cells.

A culture medium containing 1,000 to 2,000 murine P388 lymphocyticleukemia cells and test compound in predetermined concentration was putin an amount of 0.1 ml in each well of 96-well plates, and the cellswere cultured for 72 hours under a condition of 37° C. and 5% carbondioxide gas in air. After the culturing, MTT (5 mg/ml) solution wasadded in an amount of 0.02 ml in each well, and then, the cells werecultured for further 4 hours. The culture medium was centrifuged (4° C.,2,000 rpm for 20 minutes), and thus, the supernatants were removed bysuction. Then, 0.1 ml of dimethyl sulfoxide was put in each well todissolve the formed formazan, and, subsequently, another 0.1 ml ofdimethyl sulfoxide was added. The absorbance (OD) of each of theobtained solutions was measured by use of Multiskan Bichromatic (mainwavelength 570 nm, sub-wavelength 690 nm). On the supposition that theabsorbance of the untreated cells (control) was 100%, 50% proliferationinhibitory concentration (50% Inhibitory Concentration: IC₅₀ :μg/ml)were calculated by the least squares method. The results are shown inTable 1.

                                      TABLE 1                                     __________________________________________________________________________    In vitro anti-tumor activity (IC.sub.50 : μg/ml)                           against murine P388 lymphocytic leukemia cells                                        ##STR16##                IC.sub.50                                    Compound                                                                             A  R.sub.1                                                                          R.sub.3                                                                           Y'          R.sub.2                                                                           (μg/ml)                                   __________________________________________________________________________    1-1    CH H  H                                                                                  ##STR17##  CO.sub.2 H                                                                        0.0107                                       1-2    CH H  H   Same as above                                                                             CO.sub.2 H                                                                        0.00641                                                        (+) form!                                                   1-3    CH H  H   Same as above                                                                             CO.sub.2 H                                                                        0.0200                                                         (-) form!                                                   1-4    CH H  H                                                                                  ##STR18##  CO.sub.2 H                                                                        0.0200                                       2      CH H  H                                                                                  ##STR19##  CO.sub.2 H                                                                        0.0178                                       3      CH H  H                                                                                  ##STR20##  CO.sub.2 H                                                                        0.0103                                       4      CH H  H                                                                                  ##STR21##  CO.sub.2 H                                                                        0.0175                                       5      CH H  H                                                                                  ##STR22##  CO.sub.2 H                                                                        0.0117                                       6      CH H  H                                                                                  ##STR23##  CO.sub.2 H                                                                        0.0105                                       7      CH H  H                                                                                  ##STR24##  CO.sub.2 H                                                                        0.00955                                      8      CH F  H                                                                                  ##STR25##  CO.sub.2 H                                                                        0.00413                                      9      CH H  NH.sub.2                                                                           ##STR26##  CO.sub.2 H                                                                        0.00809                                      10     CH H  H                                                                                  ##STR27##  CO.sub.2 H                                                                        0.0192                                       11     CH H  H                                                                                  ##STR28##  CHO 0.0309                                       12     CH H  H                                                                                  ##STR29##  CO.sub.2 H                                                                        0.0195                                       13     CH H  H                                                                                  ##STR30##  CO.sub.2 H                                                                        0.0252                                       14     N  H  H                                                                                  ##STR31##  CO.sub.2 H                                                                        0.0466                                       15     CH H  NH.sub.2                                                                           ##STR32##  CO.sub.2 H                                                                        0.0157                                       16     CH F  H                                                                                  ##STR33##  CO.sub.2 H                                                                        0.0344                                       A      CF H  H                                                                                  ##STR34##  CO.sub.2 H                                                                        0.0942                                       H      Etoposide                 0.00849                                      __________________________________________________________________________     *The steric structure of each substituent shows relative configuration        (the same applies in the followings).                                    

Compound H: Etoposide ##STR35##

As shown in Table 1, the in vitro anti-tumor activities (IC₅₀) of thecompounds of this invention against murine P388 lymphocytic leukemiacells are 2 to 22 times stronger than that of Compound A.

Test Example 2

In vitro anti-tumor activities against human cancer cell lines

A culture medium containing 500 to 2,000 human cancer cells was put inan amount of 0.1 ml in each well of 96-well plate, and the cells werecultured for 20 hours under a condition of 37° C. and 5% carbon dioxidegas in air. After the culturing, a solution of test compounds ofpredetermined concentration was added, and then, the cells were culturedfor further 72 hours. After the culturing, an MTT (5 mg/ml) solution wasadded in an amount of 0.01 ml in the respective wells, and the cellswere cultured for further 4 hours. The supernatants of the culturemedium were removed by suction, and then, 0.1 ml of dimethyl sulfoxidewas put in respective well to dissolve the formed formazan, and,moreover, 0.1 ml of dimethyl sulfoxide was added. With regard to theresulting solution, 50% proliferation inhibitory concentrations werecalculated in the same manner as in Test example 1.

The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        In vitro anti-tumor activities (IC.sub.50 : μg/ml)                         against human cancer cell lines                                               Com-  Human Cancer Cell                                                       pound KB      HMV-2   AZ-521                                                                              MKN45 WiDr C-33A A-427                            ______________________________________                                        1-1   0.110   0.131   0.0561                                                                              0.150 0.379                                                                              0.137 0.0871                           1-2   0.0873  0.114   0.0396                                                                              0.155 0.357                                                                              0.0828                                                                              0.0925                           1-3   0.208   0.234   0.110 0.222 0.503                                                                              0.148 0.107                            1-4   0.147   0.197   0.0985                                                                              0.131 0.273                                                                              0.143 0.158                            2     0.309   0.382   0.331 0.579 1.88 0.314 0.169                            3     0.153   0.210   0.181 0.364 1.08 0.184 0.0588                           4     0.172   0.187   0.100 0.199 0.392                                                                              0.144 0.194                            5     0.0805  0.0944  0.0433                                                                              0.0978                                                                              0.218                                                                              0.106 0.0927                           6     0.0987  0.122   0.0622                                                                              0.160 0.381                                                                              0.156 0.0832                           7     0.111   0.139   0.0719                                                                              0.112 0.314                                                                              0.105 0.0933                           8     0.0494  0.0578  0.0285                                                                              0.143 0.323                                                                              0.0712                                                                              0.0625                           9     0.106   0.211   0.0625                                                                              0.148 0.412                                                                              0.151 0.100                            10    0.190   0.386   0.292 0.468 0.797                                                                              0.247 0.153                            11    0.179   0.214   0.0307                                                                              0.144 0.264                                                                              0.161 0.0749                           12    0.125   0.265   0.101 0.164 0.295                                                                              0.131 0.146                            13    0.150   0.171   0.0717                                                                              0.139 0.268                                                                              0.138 0.150                            14    0.636   0.872   0.310 --    --   --    --                               15    0.0604  0.119   0.0288                                                                              0.0964                                                                              0.259                                                                              0.0854                                                                              0.0696                           16    0.126   0.181   0.0798                                                                              0.148 0.322                                                                              0.132 0.145                            A     1.94    2.88    0.797 1.27  4.09 0.651 0.854                            H     0.201   0.298   0.080 0.490 1.63 0.084 0.095                            ______________________________________                                         KB: KB human nasopharynx cancer                                               HMV2: HMV2 human melanoma                                                     AZ521: AZ521 human stomach cancer                                             MKN45: MKN45 human stomach cancer                                             WiDr: WiDr human colorectal cancer                                            C33A: C33A human cervix cancer                                                A427: A427 human lung cancer                                             

As shown in Table 2, Compounds of this invention exhibit excellent invitro anti-tumor activities (IC₅₀) against human cancer cells. On theother hand, the activity of Compound A, i.e., the control, is only 1/2to 1/50 as high as the compounds of this invention.

Test Example 3

Increases in life span of mice to which murine P388 lymphocytic leukemiacells had been implanted

1×10⁶ murine P388 lymphocytic leukemia cells were intraperitoneallyimplanted in each of SLC: BDF1 mice (8 to 10 weeks old, female, 7animals per one group). A test compound (drug) was either dissolved in0.1 N--NaOH or suspended in 0.4% carboxymethylcellulose solution, andthe resultant solution or suspension was diluted with distilled water or0.4% carboxymethylcellulose solution so as to give predeterminedconcentrations for administration. The obtained solution wasintraperitoneally (ip) administered twice, namely on the day followingthe implantation (1st day) and the 5th day, each 0.2 ml. The life anddeath of the mice were observed over a period of 30 days, and the mediansurvival time (hereinafter referred to as MST) was determined for eachgroup, and, thus, the increase in life span (ILS; %) was calculatedaccording to the following equation:

    ILS (%)= {(MST of the test group)/(MST of the control group)}-1!×100

Drug effect was judged in the following manner, according to thecriterion of U.S. National Cancer Institute (NCI):

ILS=75% or higher: ++ (remarkably effective);

20 to 74%: + (effective);

19% or lower: - (ineffective)

The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Increases in life span of murine P388                                         lymphocytic leukemia cells implanted mice                                              Dose    ILS (%)    ILS (%)                                           Compound (mg/kg) (in solution)                                                                            (in suspension)                                                                        Evaluation                               ______________________________________                                        1-1      50      >275                ++                                                25      200                 ++                                                12.5    150        175      ++                                                6.25    125                 ++                                                3.13    100        88       ++                                                1.56    63                  +                                                 0.78    50                  +                                        1-2      25      >275                ++                                                12.5    >275       213      ++                                                6.25    175                 ++                                                3.13    125        113      ++                                                1.56    88                  ++                                                0.78    50                  +                                        1-3      50      >275       200      ++                                                25      200                 ++                                                12.5    138        125      ++                                                6.25    88                  ++                                                3.13    63         63       +                                        1-4      12.5    --         100      ++                                                3.13               25       +                                         2       50      >275       238      ++                                                25      213                 ++                                                12.5    125        138      ++                                                6.25    100                 ++                                                3.13    88         63       ++                                                1.56    50                  +                                         3       25      >275                ++                                                12.5    188        175      ++                                                6.25    113                 ++                                                3.13    75         88       ++                                                1.56    63                  +                                         4       25      150                 ++                                                12.5    125        125      ++                                                6.25    88                  ++                                                3.13    63         63       +                                         5       12.5    188        188      ++                                                6.25    150                 ++                                                3.13    113        88       ++                                                1.56    50                  +                                         6       50                 >275     ++                                                25      225                 ++                                                12.5    150        200      ++                                                6.25    138                 ++                                                3.13    100        100      ++                                                1.56    63                  +                                         7       50                 188      ++                                                12.5    --         100      ++                                                3.13               75       ++                                        8       12.5               >275     ++                                                3.13    --         175      ++                                                0.78               75       ++                                        9       50                 250      ++                                                12.5    --         125      ++                                                3.13               100      ++                                        10      50      >275                ++                                                25      125                 ++                                                12.5    100        113      ++                                                6.25    75                  ++                                                3.13    63         50       +                                         11      50                 225      ++                                                12.5    --         150      ++                                                3.13               88       ++                                        12      12.5    --         75       ++                                        13      50                 213      ++                                                12.5    --         100      ++                                                3.13               25       +                                         14      50                 >275     ++                                                12.5    --         113      ++                                                3.13               63       +                                         15      50                 238      ++                                                12.5    --         200      ++                                                3.13               88       ++                                        16      50                 238      ++                                                12.5    --         138      ++                                                3.13               38       +                                         17      50      --         150      ++                                        A       50      38                  +                                                 25      25         --       +                                                 12.5    0                   --                                        H       25      >275                ++                                                12.5    150                 ++                                                6.25    100        --       ++                                                3.13    75                  ++                                                1.56    50                  +                                        ______________________________________                                    

As shown in Table 3, the effects of the compounds of this invention onincrease in life span of mice to which murine P388 lymphocytic leukemiacells were implanted are much superior to the effects of Compound A as acontrol.

Test Example 4

Tumor growth inhibition effect on colon 26 murine tumor cells implantedmice

A 2% brei of colon 26 murine tumor cells was intracutaneously implantedeach 0.1 ml in the abdominal part of SLC: CDF₁ female mice (7 to 9 weeksold, 7 animals per one group). Then, test compound (drug) was dissolvedin 0.1 N--NaOH, and diluted with distilled water to give predeterminedconcentrations for administration. The obtained solutions wereintraperitoneally (ip) administered each 0.2 ml once a day, from the day(1st day) following the implantation to the 9th day. On the 21st or 22ndday after the implantation, tumor weight was estimated from tumordiameters, and thus, the tumor growth inhibition rate (IR %) of the drugadministered group in comparison with the control group was calculatedaccording to the following equation.

    IR (%)= 1-{(MTW in the treated group)/(MTW in the control group)}!×100

MTW: mean tumor weight

                  TABLE 4                                                         ______________________________________                                        Tumor growth inhibition rate (IR) of colon 26                                 murine tumor cells implanted mice                                             Compound       Dose (mg/kg)                                                                             IR (%)                                              ______________________________________                                        1-1            1.56       86                                                  1-2            1.10       69                                                  1-3            1.56       50                                                                 2.21       75                                                  1-4            3.13       61                                                   2             12.5       79                                                   3             3.13       77                                                   4             1.56       64                                                                 3.13       89                                                   5             1.56       78                                                   6             1.56       71                                                   7             1.56       61                                                                 3.13       95                                                   8             0.39       57                                                                 1.56       91                                                   9             3.13       62                                                                 6.25       97                                                   10            3.13       74                                                   11            0.78       50                                                                 1.56       58                                                   12            3.13       48                                                                 6.25       84                                                   13            1.56       41                                                   14            3.13       55                                                                 6.25       68                                                   15            3.13       68                                                   16            3.13       77                                                   A             12.5       23                                                   H             12.5       57                                                  ______________________________________                                    

As shown in Table 4, the tumor growth inhibition rate (IR) of thecompounds of this invention in colon 26 murine tumor cells implanted inmice are excellent. On the other hand, the effect of Compound A isapparently inferior to the compounds of this invention in view of bothdoses and inhibition rates.

In the following Test examples 5 to 9, human cancer cells were implantedin nude mice, to which test compounds dissolved in an aqueous solutioncontaining NaOH were administered, and, thus, the degree of growthinhibition of cancer cells was observed.

The results of Test examples 5 to 9 are shown in FIGS. 1-14.

FIGS. 1-9 show the variation with the lapse of days of IR.

FIGS. 10-14 show the relation between tumor weight and the lapse of daysin the case where Compound 1--1 was used in Test examples 5-9. Verticalaxis corresponds to tumor weight, and horizontal axis corresponds to thedays which passed after the start of administration. Tumor weight wasestimated from tumor diameter. The term Control in FIGS. 10-14 meanstumor weight variation with days in nude mice to which, although cancercells had been implanted, no test compounds had been administered.

Each of Figures shows the amount of each compound administered(mg/kg/day) and IR (%) on the last day of observation which is explainedin Test example 4.

In the following, there is explained schedule of administration of testcompounds in nude mice. After days of × in number have passed fromimplantation of human cancer cells in nude mice, test compounds areintraperitoneally (ip) administered for y days, followed by wash-outperiods of days of z in number. Thereafter, test compounds areadministered again for y days. In this case, the cycle of administration(y days) and discontinuation (z days) is called "course". Such aschedule is denoted by marks as follows:

(x) (y) (z) (course) (ip)!

These marks are explained below with an example. The mark "↑" means theday on which test compounds are administered.

Example: Test compounds are administered (ip) after 25 days fromimplantation, and, then, administration is suspended for six days, and,thereafter, administration is conducted again. This operation isrepeated five times. ##STR36##

Test Example 5

Anti-tumor effect on KB human nasopharynx cancer implanted in nude mice

Experiment was conducted under the following condition:

Condition:

Animal used: Female BALB/cAnNCrj-nu/nu nude mice (9 and 14 weeks old, 6animals per one group)

Cancer cells used: Human nasopharynx cancer cell KB

Implantation of cancer cells:

2.5×106 cancer cells were intracutaneously implanted at the abdominalpart of nude mice

Administration schedule:

(5)(1)(6)(6 courses)(ip)!

Results: Shown in FIGS. 1-2 and 10

Test Example 6

Anti-tumor effect on MX-1 human breast cancer cells implanted in nudemice

Experiment was conducted under the following condition:

Condition:

Animal used: Female BALB/cAnNCrj-nu/nu nude mice (9 weeks old, 5-6animals per one group)

Cancer cells used: Human breast cancer MX-1

Implantation of cancer cells:

A piece of cancer tissue of 2 mm3 was subcutaneously implanted at theback part of nude mice

Administration schedule:

(16 and 23)(1)(6)(6 courses)(ip)!

Results: Shown in FIGS. 3-5 and 11

Test Example 7

Anti-tumor effect on WiDr human colorectal cancer cells implanted innude mice

Experiment was conducted under the following condition:

Condition:

Animal used: Female BALB/cAnNCrj-nu/nu nude mice (9 weeks old, 6 animalsper one group)

Cancer cells used: Human colorectal cancer WiDr

Implantation of cancer cells:

2.5×106 cancer cells were intracutaneously implanted at the abdominalpart of nude mice

Administration schedule:

(9)(1)(6)(6 courses)(ip)!

Results: Shown in FIGS. 6 and 12

Test Example 8

Anti-tumor effect on HMV-2 human melanoma cells implanted in nude mice

Experiment was conducted under the following condition:

Condition:

Animal used: Female BALB/cAnNCrj-nu/nu and BALB/c nu/nu nude mice (11-15weeks old, 6-7 animals per one group)

Cancer cells used: Human melanoma HMV-2

Implantation of cancer cells:

4.4×106 cancer cells were intracutaneously implanted at the abdominalpart of nude mice

Administration schedule:

(8-9)(1)(6)(9 courses)(ip)!

Results: Shown in FIGS. 7-8 and 13

Test Example 9

Anti-tumor effect on LX-1 human lung cancer cells implanted in nude mice

Experiment was conducted under the following condition:

Condition:

Animal used: Female BALB/cAnNCrj-nu/nu nude mice (13 weeks old, 6animals per one group)

Cancer cells used: Human lung cancer LX-1

Implantation of cancer cells:

A piece of cancer tissue of 2 mm3 was subcutaneously implanted at theback part of nude mice

Administration schedule:

(19 and 26)(1)(6)(5-6 courses)(ip)!

Results: Shown in FIGS. 9 and 14

As is seen in the above-mentioned FIGS. 1-14, the compounds of thisinvention remarkably inhibited the growth of human cancer cellsimplanted in nude mice.

Test Example 10

Acute toxicity

Solutions of test compounds in predetermined concentrations wereadministered (0.1 ml/10 g body weight) to female BALB/c CrSlc mice (5 to10 animals per one group, 10 weeks old) respectively, and, thus, LD₅₀values were calculated from the mortalities of the mice on the 14th dayafter the administration. The results are shown in the following table.

                  TABLE 5                                                         ______________________________________                                        Acute toxicity (LD.sub.50 : mg/kg)                                                         Intraperitoneal                                                                          Intravenous                                                        administration                                                                           administration                                        Compound     (ip)       (iv)                                                  ______________________________________                                        1-1          93.2       --                                                    2            135        --                                                    3            55.2       119                                                   4            --         >100                                                  5            --         66.7                                                  H            71         123                                                   ______________________________________                                    

As is shown in Table 5, the acute toxicity of the compounds of thisinvention are almost equal to that of Compound H (Etoposide), acommercially available anti-cancer agent.

Test Example 11

Solubility

The solubilities of Compound 1-1, 1-1-3, 3 and 3-1 in 0.1M phosphatebuffer (pH 7.2) and in distilled water were measured, and the followingresults were obtained.

Compound 1-1-3 is an L-Ala derivative of Compound 1-1 and is preparedaccording to Example C-1 (5), while Compound 3-1 is an L-Ala derivativeof Compound 3 and is prepared according to Example C-6 (4).

                  TABLE 6                                                         ______________________________________                                        Solubility                                                                                            0.1 M phosphate                                                    Distilled water                                                                          buffer (pH 7.2)                                       Compound     (mg/ml)    (mg/ml)                                               ______________________________________                                        1-1          2.9        20.1                                                  1-1-3        >98.7      >233.5                                                3            8.3        --                                                    3-1          79.4       --                                                    ______________________________________                                    

As shown in Table 6, Compounds 1-1-3 and 3-1 whose Z is an amino acidresidue exhibited a solubility about 10 times or higher than that ofCompound 1-1 and 3 whose Z is a hydrogen atom. Besides, the solubilityof Compound 1-1 in neutral state is so high that it is suitable as aliquid agent such as injection.

As shown in the above test results, the compounds of this inventionexhibit remarkable anti-tumor activity against not only non-solid tumorssuch as lymphocytic leukemia tumor but also various solid tumors whichoccur in tissues of, for example, lung, breast, stomach, uterus, skin,intestine, bladder and nasopharynx. Further, the compounds of thisinvention have comparatively high safety. The compounds are thereforeuseful as agents for the treatment or prophylaxis of human tumors.

The compounds of this invention are administered in such an amount as toinhibit tumor, which amount varies depending on their pharmacodynamiccharacteristics, way of administration, symptoms and ages, objective ofadministration (prophylaxis or treatment), etc. Usually, however, thecompounds are administered in an amount of about 0.25 mg to about 50 mg,preferably about 0.5 mg to about 20 mg, per one day and per kg of bodyweight. For example, about 13 mg to about 2.5 g, preferably 25 mg to 1 gin total of active ingredient is administered per day in a patienthaving a body weight of about 50 kg. The above dose of a day may bedivided into two to four and administered separately. The administrationroute may be either oral or parenteral, but parenteral administration isrecommended.

The compounds of this invention are generally administered in the formof pharmaceutical preparations. These preparations can be prepared bycompounding the compounds of this invention with pharmaceuticalcarriers. For example, a pharmaceutical carrier for liquid agents aspharmaceutical preparations for parenteral administration contains asolvent as an essential ingredient and, if necessary, auxiliaries suchas tonicity agents, solubilizers, soothing agents, pH-adjusting agents,buffers and preservatives.

As for a solvent, there are generally employed water, organic solventsuch as propylene glycol or a mixture of water wi th organic solvent.

Examples of tonicity agent include sugars such as Sorbit and Mannit, andsodium chloride, but sugars are much preferable.

As for pH-adjusting agent, there can be employed bases such as sodiumhydroxide and acids such as hydrochloric acid and phosphoric acid.

Examples of solubilizer include surfactants such as Polysorbate 80 andPluronic F68, and organic acids such as lactic acid and methanesulfonicacid which can form an acid addition salt together with the compounds ofthis invention.

As for soothing agent, there can be employed lidocaine hydrochloride andprocaine hydrochloride. As for preservative, there can be usedbenzylalcohol, and as examples of stabilizer, there can be takenantioxidant such as ascorbic acid. As for buffer, there can be employedsalts of acid such as phosphoric acid, citric acid and lactic acid.

Liquid agents such as injections and infusions can be prepared bydissolving or suspending, preferably dissolving the compounds of thisinvention in a solvent, and by, if necessary, compounding otherauxiliaries before or after the dissolution or suspension. Lyophilizedpharmaceutical preparations can be prepared by freeze-drying theseliquids. When administered, the lyophilized pharmaceutical preparationsare re-dissolved or re-suspended.

As for carriers in solid pharmaceutical preparations such as tablets,capsules, granules, fine granules and powders, any can be used so longas they do not react with the compounds of this invention and so long asthey are used in this art. Concrete examples of such carriers includestarches, mannitol, crystalline cellulose, carboxymethylcellulose, etc.

These pharmaceutical preparations may further contain ingredients usefulfor medical treatment other than the compounds of this invention.

Preparation Processes

Processes for the preparation of the compounds of this invention are asfollows. Compounds (I) of this invention and their salts can be preparedaccording to (a) pyrrolidine substitution reaction, (b) ring closingreaction, (c) oxidation reaction, etc.

(a) Pyrrolidine substitution reaction

Among the compounds of this invention of formula (I), both compound(I-a) wherein Y is 1-pyrrolidinyl group (Y') having a substituent and asalt thereof can be prepared by making a compound having the followingformula (II) or a salt thereof ##STR37## wherein L is an eliminablegroup, and A, R₁, R₂, R₃ and m are as defined above,

react with a pyrrolidine derivative of the following formula (III)##STR38## wherein R₄, R₅, Z, n and p are as defined above.

As for the eliminable group (L) in the formula (II), there can bementioned the same groups as in the case of formula (I) where Y is aneliminable group, and preferable example of group (L) include halogenatom, lower alkoxy group, lower alkylthio group, lower alkylsulfinylgroup, lower alkylsulfonyl group, arylsulfonyl group, loweralkylsulfonyloxy group, arylsulfonyloxy group, etc.

The above reaction can be conducted either without any solvent or in asuitable solvent, and preferably in the presence of a base, at atemperature ranging from 10° to 150° C. As for the solvent, there can beused acetonitrile, water, ethanol, pyridine, dimethyl sulfoxide,1-methyl-2-pyrrolidone, etc. As for the base, there is employed such oneas functions as an acid acceptor, and concrete examples of such a baseinclude triethylamine, 1,8-diazabicyclo 5.4.0!-7-undecene, andcarbonates such as sodium carbonate and sodium bicarbonate. Compound(III) may be used in excess so that it may also act as an acid acceptor.

Compounds (II), which are used as raw materials, are also novel, and canbe prepared, for example, by the following ring closing reaction.

(b) Ring closing reaction

Compounds (I) of this invention and their salts can be prepared bysubjecting a compound represented by the following formula (IV)##STR39## wherein L is an eliminable group, and R₁, R₂, R3, A, Y and mare as defined above to ring closing reaction.

As to the eliminable group L, there can be employed the same groups asthose included in the definition of Y, as is explained in the above withregard to formula (II).

This ring closing reaction can be conducted by stirring a mixture ofCompound (IV) with a solvent in the presence of a base such as potassiumcarbonate, sodium carbonate, sodium hydride, potassium t-butoxide orpotassium fluoride, the amount of which base is one to three times inmole as much as Compound (IV), at a temperature ranging from 30° to 150°C., preferably 30° to 100° C., for 1 to 6 hours. Examples of suitablesolvent include ethanol, dioxane, tetrahydrofuran, dimethylformamide,dimethyl sulfoxide, etc.

Compounds (IV), which are used as raw materials, are also novel, and canbe prepared according to the processes in Examples which are describedlater.

(c) Oxidation reaction

Compounds of this invention represented by formula (I) and sal tsthereof can be prepared by subjecting a compound represented by thefollowing formula (V) ##STR40## wherein R₁, R₃, A, Y and m are asdefined above to an oxidation reaction.

This oxidation reaction is carried out by mixing the above Compound (V)with an oxidizer in a solvent and stirring the resulting mixture forseveral hours at a temperature of 100° C. or lower, preferably 0° to 50°C. Examples of the oxidizer include2,3-dichloro-5,6-dicyanobenzoquinone, tetrachloro-1,4-benzoquinone,tetracyanoethylene, palladium-carbon, N-bromosuccinimide and manganesedioxide. As for examples of the solvent, there can be taken 1,4-dioxane,toluene, xylene, ethanol, t-butanol, ethyl acetate, dimethylformamide,etc.

Compounds (I) of this invention and salts thereof can also be preparedby making Compound (I) of this invention wherein Z is a hydrogen atomreact with amino acid or peptide according to a usual method, or byaminating Compound (I) wherein R₃ is a halogen atom so as to convertsaid compound into a compound wherein R₃ is an amino group.

When the compounds of this invention obtained in the above-mentionedmanner are ester forms, said compounds can be converted into carboxylicacid forms by means of hydrolyzing the ester parts by a usual method.Further, the carboxylic acid parts of Compounds (I) can also beesterified by a usual method. Furthermore, when Z of the compounds ofthis invention is an amino acid residue or peptide residue protected bya protecting group, said protecting group can be eliminated by a usualmethod.

The compounds of this invention thus prepared can be isolated andpurified by usual processes. According to the conditions of isolationand purification, these compounds are obtained in the form of salts,free carboxylic acids or free amines, which are then converted from oneinto another in accordance with purposes, and, thus, there can beobtained compounds in desired forms.

When the compounds of this invention are racemic, they can, ifnecessary, be separated into respective optical isomers by knownmethods. The stereoisomers (cis form and trans form) of the compounds ofthis invention can, if necessary, be separated from one anotheraccording to a usual method such as, for example, a fractionalcrystallization method or a chromatography method.

It is of course possible to use an optical isomer or a stereoisomer as araw material and lead it to a desired substance corresponding thereto,and such a method is generally advantageous.

Examples

This invention is detailedly explained by working examples below.

In the following, Examples of Series A include specific examples ofprocesses for the preparation of intermediates (II) general formula(II)!, Examples of Series B include specific examples of processes forthe preparation of raw materials (III) general formula (III)!, Examplesof Series C include specific examples of processes for the preparationof object substances (I-a), and Examples of Series D include specificexamples of processes for the preparation of pharmaceuticalpreparations.

Further, the attached FIGS. 15-19 show reaction formulae of thereactions which are mentioned in the following Examples of Series A,Series B and Series C-14.

1. Series A

Example A-1 Preparation of intermediate (II)7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester

(1)-1 2,6-Dichloropyridine (20 g) was dissolved in tetrahydrofuran (200ml), and, to the resulting solution, there was added dropwise a solutionof n-butylithium (1.6M) in n-hexane (84.5 ml) in a flow of argon gas ata temperature of -78° C. over a period of 30 minutes. After theresulting solution was stirred for one hour at the same temperature,greatly excess carbon dioxide (solid) was added. After stirring for onehour, temperature was raised to -10° C., and, then, water andhydrochloric acid were added in order so that pH might be 1-2, and,thereafter, the resultant solution was extracted with ethyl acetate.After the obtained extract was dried over anhydrous sodium sulfate,solvent was distilled off under reduced pressure to dryness. To theobtained solid, there was added thionyl chloride (40 ml), and theresulting mixture was heated to reflux for three hours. The excessthionyl chloride was distilled off under reduced pressure, and, then,crude product was distilled under reduced pressure to form2,6-dichloronicotinoyl chloride (19.8 g).

Boiling point: 97°-99° C./1 mmHg

IR (neat) cm⁻¹ : 1784

(1)-2 A mixture of 2,6-dichloronicotinic acid (50.6 g) with thionylchloride (100 ml) was refluxed for 2 hours. The excess thionyl chloridewas distilled off under reduced pressure, and crude product wasdistilled under reduced pressure to give 2,6-dichloronicotinoyl chloride(44.9 g).

Boiling point: 115°-120° C./3 mmHg

IR (neat) cm⁻¹ : 1784

(2) Ethoxymagnesiummalonic acid diethyl ester derived from metalmagnesium (5.36 g), malonic acid diethyl ester (35.4 g) and ethanol (27ml) was dissolved in a mixed solution composed of tetrahydrofuran (35ml) and toluene (140 ml). The resultant solution was cooled with ice,and, to this solution under stirring, there was added dropwise a mixedsolution composed of tetrahydrofuran (19 ml) and toluene (32 ml)containing the acid chloride (44.9 g) obtained in the above (1)-1 or(1)-2. After completion of the dropwise addition, the obtained mixturewas stirred overnight at room temperature. The mixture was concentratedunder reduced pressure, and aqueous solution of hydrochloric acid wasadded to the residue, and, then, the mixture was extracted with ethylacetate. The obtained extract was dried over anhydrous sodium sulfate,and the solvent was distilled off under reduced pressure, and, thus,there was produced an oily material. Water (190 ml) andp-toluenesulfonic acid (0.1 g) were added to the oily material, and themixture was refluxed for 2 hours. After cooled, the mixture wasextracted with chloroform and dried over anhydrous sodium sulfate, andthen, the solvent was distilled off under reduced pressure to give anoily material. The obtained crude product was distilled under reducedpressure to produce 2,6-dichloronicotinoylacetic acid ethyl ester (45.2g).

Boiling point: 135°-140° C./2 mmHg

(3) A mixture of the compound (44.9 g) obtained in the above (2), aceticanhydride (43.8 g) and ethyl orthoformate (37.7 g) was refluxed for onehour. The mixture was concentrated to dryness under reduced pressure,and, under ice cooling, diisopropyl ether (500 ml) and 2-aminothiazole(20 g) were added, and then, the resulting mixture was stirred at roomtemperature for 5 hours. The crystals were taken by filtration to give2-(2,6-dichloronicotinoyl )-3-(2-thiazolyl-amino)acrylic acid ethylester (52.8 g).

Melting point: 119°-122° C. (recrystallized from diisopropyl ether)

IR (KBr) cm⁻¹ : 1700

(4) The compound (51.7 g) obtained in the above (3) was dissolved indioxane (310 ml), and, then, potassium carbonate (21.4 g) was added, andthe resulting mixture was stirred at 60° C. for one hour. After icewater was added, the mixture was neutralized with 10% aqueous solutionof hydrochloric acid, and the crystals were taken by filtration. Thecrystal s were recrystallized from a mixed solution composed ofchloroform and diisopropyl ether to give7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (44.6 g).

Melting point: 176°-177° C.

IR (KBr) cm⁻¹ : 1724

NMR (CDCl₃) δ: 1.43 (t, 3H, J=6.5 Hz), 4.45 (q, 2H, J=6.5 Hz), 7.38 (d,1H, J=3.5 Hz), 7.52 (d, 1H, J=8.5 Hz), 7.75 (d, 1H, J=3.5 Hz), 8.78 (d,1H, J=8.5 Hz), 10.00 (s, 1H)

Example A-2 Preparation of intermediate (II)7-chloro-1-(4-fluoro-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid ethyl ester

A mixture composed of the ester (250 mg) obtained in Example 1 (4),N-fluoro-2,6-dichloro-pyridinium tetrafluoroborate (240 mg) and1,2-dichloroethane (10 ml) was heated to reflux for two days.

Water was added to the reaction solution, and the resultant solution wasextracted with chloroform. After the obtained extract was dried overanhydrous sodium sulfate, solvent was distilled off under reducedpressure. The obtained residue was purified by silica gel columnchromatography (eluent: chloroform), and the above-identified compound(40 mg) was obtained by recrystallization from ethyl acetate.

Melting point: 174°-175° C.

IR (KBr) cm⁻¹ : 1700

NMR (CDCl₃) δ: 1.42 (t, 3H, J=6.5 Hz), 4.45 (q, 2H, J=6.5 Hz), 7.33 (d,1H, J=3.5 Hz), 7.51 (d, 1H, J=8.5 Hz), 8.78 (d, 1H, J=8.5 Hz), 9.85 (s,1H)

Example A-3 Preparation of intermediate (II)5,7-dichloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

(1) To a mixture composed of 4-amino-2,6-dichloropyridine (5.5 g),cuprous chloride (4.4 g) and concentrated hydrochloric acid (50 ml),sodium nitrite (3.5 g) was added little by little under cooling with iceand sodium chloride. After the resultant mixture was stirred for onehour at the same temperature and for one and a half hour at a roomtemperature, water was added, and the mixture was extracted withchloroform. After the obtained extract was dried over anhydrous sodiumsulfate, solvent was distilled off under reduced pressure to give2,4,6-trichloropyridine (5.5 g).

IR (neat) cm⁻¹ : 1563, 1357, 1155, 851, 823

NMR (CDCl₃) δ: 7.31 (s, 2H)

(2) To a mixture composed of the compound (5.5 g) obtained in the above(1) and tetrahydrofuran (55 ml), there was added dropwise a solution ofn-butylithium (1.6M) in n-hexane (20 ml) at a temperature of -78° C.After the resulting solution was stirred for one hour at the sametemperature, greatly excess carbon dioxide (solid) was added. Afterstirring for one hour, temperature was raised to 0° C., and, then, anaqueous solution of hydrochloric acid was added so that the solutionmight be acidic, and, thereafter, the resultant solution was extractedwith ethyl acetate. After the obtained extract was dried over anhydroussodium sulfate, solvent was distilled off under reduced pressure. To theobtained residue, there was added diisopropyl ether, and crystals weretaken by filtration, and, thus, there was obtained2,4,6-trichloronicotinic acid (6.5 g).

Melting point: 138°-141° C.

IR (KBr) cm⁻¹ : 1715

(3) A mixture of the compound (6.5 g) obtained in the above (2) withthionyl chloride (25 ml) was refluxed for three hours. Excessive thionylchloride was distilled off under reduced pressure, and crude product wasdistilled under reduced pressure to give 2,4,6-trichloronicotinoylchloride (6.6 g).

Boiling point: 93°-95° C./1 mmHg

IR (neat) cm⁻¹ : 1791

(4) A solution of methylmagnesium bromide (3M) dissolved in ether (19ml) was added dropwise at a temperature of 0° C. to a mixture composedof malonic acid monoethyl ester (3.6 g) and tetrahydrofuran (30 ml).After the resultant solution was stirred for one hour at roomtemperature, a mixture composed of the compound (6.6 g) obtained in theabove (3) and tetrahydrofuran (30 ml) was added dropwise, and theresulting solution was heated at 60° C. for one hour and a half. Solventwas distilled off under reduced pressure, and aqueous solution ofhydrochloric acid was added to the obtained residue, and, then, themixture was extracted with chloroform. The obtained extract was driedover anhydrous sodium sulfate, and the solvent was distilled off underreduced pressure, and, then, the obtained crude product was distilledunder reduced pressure to produce 2,4,6-trichloro-nicotinoylacetic acidethyl ester (4.8 g).

Boiling point: 160°-162° C./2 mmHg

IR (neat) cm⁻¹ : 1746

(5) A mixture of the compound (4.8 g) obtained in the above (4), aceticanhydride (4.2 g) and ethyl orthoformate (3.6 g) was refluxed for onehour and a half. The mixture was concentrated to dryness under reducedpressure, and, under ice cool ing, diisopropyl ether (100 ml) and2-aminothiazole (1.6 g) were added, and then, the resulting mixture wasstirred at room temperature for three hours. Solvent was distilled offunder reduced pressure, and the obtained residue was purified by silicagel column chromatography (eluent: chloroform), and, thus,2-(2,4,6-trichloronicotinoyl)-3-(2-thiazolyl-amino)acrylic acid ethylester (4.0 g) was produced by recrystallization from ethyl acetate.

Melting point: 126°-127° C.

IR (KBr) cm⁻¹ : 1691

(6) A mixture composed of the compound (4.0 g) obtained in the above(5), potassium carbonate (1.5 g) and ethyl acetate (40 ml) was heated at60° C. for one hour. Solvent was distil led off under reduced pressure,and water was added to the obtained residue, and, then, the resultantmixture was extracted with chloroform. The obtained extract was driedover anhydrous sodium sulfate, and the solvent was distilled off underreduced pressure, and, then, the obtained residue was purified by silicagel column chromatography (eluent: chloroform), and, thus,5,7-dichloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (2.5 g) was obtained by recrystallization fromchloroform.

Melting point: 226°-227° C.

IR (KBr) cm⁻¹ : 1737, 1692

(7) A mixture of the ester (1.8 g) obtained in the above (6) with 20%aqueous solution of hydrochloric acid (60 ml) was heated to reflux forfive hours. After the mixture was cooled, water was added, and crystalswere taken by filtration, and then, the crystals were washed with waterto give the above-identified5,7-dichloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid (1.4 g).

Melting point: 264°-266° C.

IR (KBr) cm⁻¹ : 1729

Example A-4 Preparation of intermediate (II)5-amino-7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid

(1) A mixture of the compound (500 mg) obtained in Example A-3 (6),benzylamine (140 mg), triethylamine (280 mg) and toluene (15 ml) wasrefluxed for 30 minutes. Solvent was distilled off under reducedpressure, and water was added to the obtained residue, and, then, theresultant mixtu re was extracted with chloroform. The obtained extractwas dried with anhydrous sodium sulfate, and then, the solvent wasdistilled off under reduced pressure. The obtained residue was subjectedto recrystallization from ethyl acetate, and, thus,5-benzylamino-7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (510 mg) was produced.

Melting point: 141°-143° C.

IR (KBr) cm⁻¹ : 1733

NMR (CDCl₃) δ: 1.42 (t, 3H, J=7 Hz), 4.41 (q, 2H, J=7 Hz), 4.49 (d, 2H,J=6.5 Hz), 6.47 (s, 1H), 7.31 (d, 1H, J=3.5 Hz), 7.32-7.40 (m, 5H), 7.70(d, 1H, J=3.5 Hz), 9.87 (s, 1H), 11.2-11.7 (m, 1H)

(2) A mixture composed of the ester (1.0 g) obtained in the above (1),concentrated sulfuric acid (2 ml) and acetic acid (8 ml) was stirred at110° C. for five hours. After cooling, 8 ml of water was added, andthen, the mixture was sti rred at 110° C. for one hour. Crystals weretaken out by filtration and washed with water to give 740 mg of theabove-identified compound.

Melting point: 264°-265° C.

IR (KBr) cm⁻¹ : 1727

Example A-5 Preparation of intermediate (II)7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-5-trifluoromethyl-1,8-naphthyridine-3-carboxylicacid ethyl ester

(1) A mixture of 2,6-dichloro-4-trifluoromethylpyridine (5 g) withtetrahydrofuran (50 ml) was cooled to -78° C., and a solution ofn-butylithium (1.6M) in n-hexane (16 ml) was added dropwise to themixture, which was then stirred for 30 minutes. Then, greatly excessivecarbon dioxide (solid) was added to this mixture, which was subsequentlystirred for one hour. After the temperature was raised up to 0° C., themixture was extracted with ethyl acetate and dilute hydrochloric acid,and the resultant orgnaic layer was dried over sodium sulfate. Solventwas distilled off under reduced pressure, and thionyl chloride (20 ml)was added to the obtained residue, and then, the resulting mixture washeated to reflux for six hours. Excess thionyl chloride was distilledoff under reduced pressure, and then, the obtained residue was distilledunder reduced pressure to give 2,6-dichloro-4-trifluoromethylnicotinoylchloride (3.8 g).

Boiling point: 77°-78° C./2 mmHg

IR (neat) cm⁻¹ : 1797

(2) A drop of carbon tetrachloride was added to a mixture of magnesium(0.36 g) with ethanol (1.5 ml), and then, a mixture composed of diethylmalonate (2.4 g), ethanol (1.5 ml) and toluene (10 ml) was addeddropwise to the above mixture, which was subsequently stirred for twohours. After the mixture was cooled with ice, a mixture composed of thecompound (3.8 g) obtained in the above (1) and tetrahydrofuran (10 ml)was added dropwise to the mixture, which was then stirred for threehours at room temperature. Then, the mixture was extracted with ethylacetate and dilute hydrochloric acid, and the resultant orgnaic layerwas dried over sodium sulfate. Solvent was distilled off under reducedpressure, and water (20 ml) and p-toluenesulfonic acid (50 mg) wereadded to the obtained residue, and then, the resulting mixture washeated to reflux for three hours. The mixture was subsequently extractedwith chloroform and water, and the resultant orgnaic layer was driedover sodium sulfate. Solvent was distilled off under reduced pressure togive 2,6-dichloro-4-trifluoromethyl-nicotinoyl acetic acid ethyl ester(0.9 g).

IR (neat) cm⁻¹ : 1744, 1721

MS (m/z): 330 (MH⁺)

(3) A mixture composed of the compound (0.9 g) obtained in the above(2), ethyl orthoformate (0.6 g) and acetic anhydride (0.7 g) was heatedto reflux for 1.5 hours at 140° C., and then was concentrated to drynessunder reduced pressure. Isopropyl ether (20 ml) was added to theobtained residue, and then, under ice cooling, 2-aminothiazol (0.3 g)was added. After stirring for three hours at room temperature, solventwas distilled off. To this residue, chloroform and water were added forextraction, and the resultant orgnaic layer was dried over sodiumsulfate. Solvent was distilled off under reduced pressure, and theobtained residue was purified by silica gel column chromatography(eluent: chloroform) to give2-(2,6-dichloro-4-trifluoromethylnicotinoyl)-3-(2-thiazolylamino)acrylicacid ethyl ester (0.37 g).

IR (neat) cm⁻¹ : 1713

MS (m/z): 440 (MH⁺)

(4) A mixture composed of the compound (0.37 g) obtained in the above(3), potassium carbonate (0.13 g) and ethyl acetate (10 ml) was heatedto reflux for 15 minutes. Ethyl acetate and water were added to themixture for extraction, and the resultant orgnaic layer was dried oversodium sulfate. Solvent was distilled off under reduced pressure, andthe obtained residue was subjected to recrystallization from ethylacetate, and thus, the above-identified7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-5-trifluoromethyl-1,8-naphthyridine-3-carboxylic acid ethyl ester (0.27 g) was obtained.

Melting point: 184°-185° C.

IR (KBr) cm⁻¹ : 1736, 1703

Example A-6 Preparation of intermediate (II)5,8-dihydro-2-methanesulfonyl-5-oxo-8-(2-thiazolyl)pyrido2,3-d!pyrimidine-6-carboxylic acid ethyl ester

(1) A solution of malonic acid monoethyl ester (12.3 g) dissolved intetrahydrofuran (80 ml) was cooled with ice, and then, to the resultingsolution, a solution of methylmagnesium bromide (3M) in ether (64 ml)was added dropwise. After the resultant mixture was stirred for 20minutes, a solution of2-methylthio-4-chloropyrimidine-5-carbonylchloride (8.6 g) dissolved intetrahydrofuran (100 ml) was added dropwise to the mixture, which wasthen stirred for two hours at a room temperature. This reaction mixturewas poured into ice water, and then, concentrated hydrochloric acid wasadded to the resulting solution so that pH might be adjusted to be 5-6,and the solution was subsequently extracted with ethyl acetate. Theobtained extract was dried over anhydrous sodium sulfate, and solventwas distilled off under reduced pressure. Then, purification wasconducted by silica gel column chromatography (eluent: chloroform), andthus, 3-(2-methylthio-4-chloropyrimidin-5-yl)-3-oxopropionic acid ethylester (8.0 g) was obtained.

IR (neat) cm⁻¹ : 1743

MS (m/z): 275 (MH⁺)

(2) A mixture composed of the compound (7.95 g) obtained in the above(1), orthoethyl formate (6.80 g) and acetic anhydride (7.76 g) washeated to reflux at 130° C. for one hour, and then was concentratedunder reduced pressure. Under ice cooling, diisopropyl ether (100 ml)and 2-aminothiazol (3.28 g) were added to the mixture, which was thenstirred over night at room temperature. Crystal was taken by filtrationand then was washed with diisopropyl ether. This crystal was dissolvedin 1,4-dioxane (70 ml), and, to the resulting solution under icecooling, potassium carbonate (2.72 g) was added, and the resultingmixture was stirred at room temperature for five hours. This mixture wascooled with ice, and ice water (200 ml) was added to the mixture, whichwas then neutralized with 10% aqueous solution of hydrochloric acid.Crystal was taken by filtration, and the crystal was washed with water,1,4-dioxane and diisopropyl ether successively, and, thus,5,8-dihydro-2-methylthio-5-oxo-8-(2-thiazolyl)pyrido2,3-d!pyrimidine-6-carboxylic acid ethyl ester (6.0 g) was obtained.

Melting point: 183°-184° C.

IR (KBr) cm⁻¹ : 1736

(3) A solution of the compound (5.99 g) obtained in the above (2)dissolved in methylene chloride (450 ml) was cooled with ice, and, tothe resulting mixture, 80% m-chloroperbenzoic acid (9.30 g) was addedlittle by little, and the resultant mixture was stirred over night atroom temperature. The mixture was then washed with an aqueous solutionof sodium thiosulfate, an aqueous solution of sodium hydrogencarbonateand a saturated aqueous solution of sodium chloride successively. Afterthe mixture was dried over sodium sulfate, solvent was distilled offunder reduced pressure. Then, the above-identified5,8-dihydro-2-methanesulfonyl-5-oxo-8-(2-thiazolyl)pyrido2,3-d!pyrimidine-6-carboxylic acid ethyl ester (4.48 g) was obtained byrecrystallization from a mixed solution composed of ethyl acetate anddiisopropylether.

Melting point: 185°-187° C.

IR (KBr) cm⁻¹ : 1741

2. Series B

Example B-1 Preparation of raw material compound (III)Trans-3-(N-t-butoxycarbonylmethylamino)-4-methylpyrrolidine

(1) Trans-3-amino-1-benzyl-4-methylpyrrolidine (19 g) was dissolved inmethylene chloride (200 ml), and, to the resulting solution, a solutionof di-t-butyl dicarbonate (22.9 g) in methylene chloride (20 ml) wasadded under ice cooling. The resultant mixture was stirred at roomtemperature for one hour. Then, this reaction solution was concentratedunder reduced pressure to givetrans-1-benzyl-3-(t-butoxycarbonylamino)-4-methylpyrrolidine (28.2 g)

Melting point: 138°-140° C. (recrystallized from ethyl acetate-n-hexane)

IR (KBr)cm⁻¹ : 3198, 1706

MS (m/z): 291 (MH⁺)

(2) A 70% toluene solution (40 ml) of sodiumbis(2-methoxyethoxy)aluminum hydride was dissolved in 150 ml of toluene,and, to the resulting solution, the compound (10 g) obtained in theabove (1) was added little by little under ice cooling. The obtainedreaction mixture was heated to reflux for one hour, and, after icecooling, excess reagent was decomposed with water. Then, insolublesubstance was separated by filtration, and the obtained filtrate wasdried over anhydrous magnesium sulfate, and solvent was distilled offunder reduced pressure. The resultant residue was dissolved in methylenechloride (100 ml), and to the resulting solution, there was added underice-cooling a solution of di-t-butyl dicarbonate (7.5 g) in methylenechloride (10 ml). The obtained mixture was stirred at room temperaturefor 3.5 hours, and then, was concentrated under reduced pressure. Theresultant residue was purified by silica gel column chromatography(eluent n-hexane:ethyl acetate=5:1) to givetrans-1-benzyl-3-(N-t-butoxycarbonylmethylamino)-4-methyl-pyrrolidine(9.9 g)

IR (neat)cm⁻¹ : 1694

MS (m/z): 305 (MH⁺)

(3) The compound (1.52 g) obtained in the above (2) was dissolved inethanol (50 ml), and, to the resulting solution, there was added 10%palladium carbon (200 mg), and was absorbed a theoretical amount ofhydrogen at 50° C. After the catalyst was separated by filtration,solvent was distilled off under reduced pressure, and thus,trans-3-(N-t-butoxycarbonylmethylamino)-4-methylpyrrolidine (950 mg) wasobtained.

IR (neat)cm⁻¹ : 3337, 1685

MS (m/z): 215 (MH⁺)

Example B-2 Preparation of raw material compound (III)(+)-Trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidine

(1) Trans-3-amino-1-benzyl-4-methoxypyrrolidine (racemic; 22.4 g)disclosed in Japanese Laid-open Patent Publication No. 69474/1990 and19.6 g of L-tartaric acid were dissolved in methanol (350 ml), and theresulting solution was left still at room temperature for 7 hours.Precipitated L-tartrate was taken by filtration, and then, was subjectedto recrystallization with methanol and water, and thus, there wasobtained trans-3-amino-1-benzyl-4-methoxypyrrolidine L-tartrate (14.1 g)having the following physical properties. Then, all the mother liquorswere put together, and the solvent was distilled off under reducedpressure, and then, a saturated brine was added, and, subsequently,potassium carbonate was added so that the obtained mixture might becomebasic. Then, the mixture was extracted with ethyl acetate. The obtainedextract was washed with saturated brine, and was then dried overanhydrous sodium sulfate, and solvent was distilled off under reducedpressure. The resultant residue and D-tartaric acid (6.73 g) weredissolved in methanol (180 ml), and the obtained solution was left stillat room temperature for 7 hours. Precipitated D-tartrate was taken byfiltration, and was then subjected to recrystallization with methanoland water, and thus, there was obtainedtrans-3-amino-1-benzyl-4-methoxypyrrolidine D-tartrate (9.9 g) havingthe following physical properties.

L-tartrate

Melting point: 206°-208° C. (decomposed)

a!_(D) ²⁹ +33.0° (c=1.003, water)

Elementary analysis (%): as C₁₂ H₁₈ N₂ O.3/2 C₄ H₆ O₆

Calculated value: C, 50.11; H, 6.31; N, 6.49

Found value: C, 49.85; H, 6.26; N, 6.27 D-tartrate

Melting point: 207°-209° C. (decomposed)

a!_(D) ²⁹ -33.4° (c=1.020, water)

Elementary analysis (%): as C₁₂ H₁₈ N₂ O.3/2C₄ H₆ O₆

Calculated value: C, 50.11; H, 6.31; N, 6.49

Found value: C, 50.35; H, 6.32; N, 6.47

(2) Saturated brine was added to the L-tartrate (3.65 g) obtained in theabove (1), and the resulting mixture was neutralized with potassiumcarbonate, and was then extracted with ethyl acetate. The obtainedextract was washed with saturated brine, and was subsequently dried overanhydrous sodium sulfate. Then, solvent was distilled off under reducedpressure, and thus, there was obtained(+)-trans-3-amino-1-benzyl-4-methoxypyrrolidine (1.23 g).

a!_(D) ²⁷ +32.2° (c=1.053, methanol)

(3) The compound (5.74 g) obtained in the above (2) was dissolved inmethanol (65 ml), and, under ice cooling, di-t-butyl dicarbonate (7.29g) was added to the resulting solution, which was then stirred at thesame temperature for 30 minutes, and at room temperature for 4 hours.Solvent was distilled off under reduced pressure, and the resultantresidue was purified by silica gel column chromatography (eluentchloroform:methanol=50:1), and thus, there was obtained(+)-trans-1-benzyl-3-(t-butoxycarbonylamino)-4-methoxypyrrolidine (8.55g).

Melting point: 44°-45° C.

a!_(D) ²⁹ +9.5° (c=1.044, methanol)

(4) Lithium aluminum hydride (3.43 g) was suspended in anhydroustetrahydrofuran (150 ml), and, to the resulting suspension, an anhydroustetrahydrofuran solution (50 ml) of the compound (8.4 g) obtained in theabove (3) was added dropwise, and the resultant mixture was stirred atroom temperature for one hour. After the mixture was refluxed for 5hours, excess reagent was decomposed with water under ice cooling, andthen, insoluble substance was removed by filtration. Then, the obtainedfiltrate was extracted with ethyl acetate. The resultant extract waswashed with saturated brine, and was then dried over anhydrous sodiumsulfate. Solvent was distilled off under reduced pressure, and theobtained residue was dissolved in methylene chloride (180 ml), and, tothe resultant solution, di-t-butyl dicarbonate (6.3 g) was added underice cooling. The resultant mixture was stirred at the same temperaturefor 30 minutes, and at room temperature for 2 hours, and then, solventwas distilled off under reduced pressure. The resultant residue waspurified by silica gel column chromatography (eluentchloroform:methanol=50:1), and thus, there was obtained(+)-trans-1-benzyl-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidine(8.26 g).

a!_(D) ²⁹ +9.9° (c=1.002, methanol)

(5) The desired(+)-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidine (5.59g) was obtained from the compound (8.15 g) produced in the above (4), inthe same manner as in Example B-1 (3).

a!_(D) ²⁹ +12.5° (c=1.051, methanol)

IR (neat) cm⁻¹ : 3318, 1693

MS (m/z): 231 (MH⁺)

Example B-3 Preparation of raw material compound (III)(-)-Trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidine

(1) (-)-Trans-3-amino-1-benzyl-4-methoxypyrrolidine (1.01 g) wasobtained from the D-tartrate (2.57 g) produced in Example B-2 (1), inthe same manner as in Example B-2 (2).

a!_(D) ²⁷ -32.7° (c=1.016, methanol)

(2) (-)-Trans-1-benzyl-3-(t-butoxycarbonylamino)-4-methoxypyrrolidine(4.5 g) was obtained from the compound (3.03 g) produced in the above(1), in the same manner as in Example B-2 (3).

Melting point: 44-45° C.

a!_(D) ²⁹ -9.5° (c=1.080, methanol)

(3)(-)-Trans-1-benzyl-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidine(4.25 g) was obtained from the compound (4.25 g) produced in the above(2), in the same manner as in Example B-2 (4).

a!_(D) ²⁹ -10.1° (c=1.054, methanol)

(4) The desired(-)-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidine (2.81g) was obtained from the compound (4.25 g) produced in the above (3), inthe same manner as in Example B-2 (5).

a!_(D) ²⁹ -12.2° (c=1.003, methanol)

IR (neat) cm⁻¹ : 3318, 1693

MS (m/z): 231 (MH⁺)

Example B-4 Preparation of raw material compound (III)3-(N-t-butoxycarbonylmethylamino)-3-methylpyrrolidine

(1) 1-Benzyl-3-(N-t-butoxycarbonylamino)-3-methylpyrrolidine (28.3 g)was obtained from 3-amino-1-benzyl-3-methylpyrrolidine (20 g) in thesame manner as in Example B-1 (1).

IR (neat) cm⁻¹ : 3356, 1716, 1697

MS (m/z): 291 (MH⁺)

(2) 1-Benzyl-3-(N-t-butoxycarbonylmethylamino)-3-methylpyrrolidine (7.5g) was obtained from the compound (11.4 g) produced in the above (1), inthe same manner as in Example B-1 (2).

IR (neat) cm⁻¹ : 1697

MS (m/z): 305 (MH⁺)

(3) The desired 3-(N-t-butoxycarbonylmethylamino)-3-methylpyrrolidine(5.5 g) was obtained from the compound (7.5 g) produced in the above(2), in the same manner as in Example B-1 (3).

IR (neat) cm⁻¹ : 3337, 1682

MS (m/z): 215 (MH⁺)

3. Series C

Example C-1 Preparation of desired product (I)1,4-Dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid, its salt and its L-Ala derivative

(1) Triethylamine (18 ml) was added to a suspension composed of7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (7.1 g) obtained in Example A-1 (4),trans-3-methoxy-4-methylaminopyrrolidine dihydrochloride (6.0 g) andacetonitrile (150 ml). The resulting reaction mixture was stirred atroom temperature for 5 hours, and was then concentrated under reducedpressure. Then, an aqueous solution of sodium hydrogencarbonate wasadded, and the resultant mixture was extracted with chloroform. Afterthe obtained extract was dried over anhydrous sodium sulfate, solventwas distilled off under reduced pressure, and the resultant residue waspurified by silica gel column chromatography (eluentchloroform:methanol=6:1), and thus, there was obtained1,4-dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (6.1 g).

Melting point: 73°-76° C.

(2) A solution composed of the above ester (6.0 g) nd an 18% aqueoussolution of hydrochloric acid (100 ml) was stirred at 100° C. for 28hours. Crystals were taken by filtration, and washed with a mixedsolution composed of ethanol and diisopropyl ether to give1,4-dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 1-1)(4.45 g).

Melting point: 270°-273° C.

(3) A solution composed of the hydrochloride (51.5 g) obtained in theabove (2), water (500 ml) and aqueous ammonia (40 ml) was stirredovernight at 50° C. Acetonitrile was added to this solution, which wasthen concentrated under reduced pressure, and crystals were taken byfiltration. The crystals were washed with water and acetonitrile to give1,4-dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid (Compound 1-1-1)(32.6 g).

Melting point: 290°-292° C. (decomposed)

(4) A mixture composed of the compound (2.0 g) obtained in the above(3), lactic acid (3.1 g) and distilled water (4 ml) was heated at 60° C.to be dissolved. After the resultant solution was cooled to roomtemperature, ethanol (70 ml) was added, and crystals were taken byfiltration and washed with ethanol to give 2 g of a lactate (Compound1-1-2).

Melting point: 288°-291° C. (decomposed)

(5) To a mixture composed of the ester (1.89 g) obtained in the above(1), N-t-butoxycarbonyl-L-alanine (1.26 g) and methylene chloride (80ml), there was added 1.27 g of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC), andthe resultant mixture was stirred at room temperature for 3 hours. Afterwashed with water, the mixture was dried over anhydrous sodium sulfate,and was then concentrated under reduced pressure. The resultingconcentrate was purified by silica gel column chromatography (eluentchloroform:methanol=50:1) to give 7-{trans-3-N-(N-t-butoxycarbonyl-L-alanyl)!methylamino-4-methoxy-1-pyrrolidinyl}-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (2.15 g).

Melting point: 120°-123° C.

a!_(D) ²⁸ +10.2° (c=1.0, chloroform)

A mixture composed of this ethyl ester (1.71 g), 0.5N hydrochloric acid(42 ml) and ethanol (22 ml) was stirred under heating at 80° C. for 17.5hours. The resultant solution was concentrated under reduced pressure,and crystals were taken by filtration, and were then washed with 10%hydro-chloric acid and ethanol to give 1.16 g of 7-trans-3-(N-L-alanylmethylamino)-4-methoxy-1-pyrrolidinyl!-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 1-1-3).

Melting point: 230°-233° C.

a!_(D) ²⁹ +8.40 (c=1.0, water)

Example C-2 Preparation of desired compound (I)(+)-1,4-Dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid (Compound 1-2-1) and its hydrochlorid (Compound 1-2)

(1) With use of7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester obtained in Example A-1 (4) and(+)-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidineobtained in Example B-2, there was produced (-)-7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (amorphous) in the same manner as in Example C-1 (1).

a!_(D) ²⁹ -9.1° (c=1.006, chloroform)

(2) The above-captioned hydrochloride (Compound 1-2) was obtained fromthe ethyl ester obtained in the above (1), according to the processdescribed in Example C-1 (2).

Melting point: 278°-282° C. (decomposed)

a!_(D) ²⁹ +24.8° (c=0.500, water)

(3) A solution composed of the hydrochloride (28.1 g) obtained in theabove (2), water (300 ml) and aqueous ammonia (25 ml) was stirredovernight at 50° C., and was then concentrated under reduced pressure.Crystals were washed with water and methanol to give the desiredcarboxylic acid (Compound 1-2-1) (19.5 g).

Melting point: 268°-271° C.

a!_(D) ³⁰ +53.1° (c=1.005, 1N NaOH)

Example C-3 Preparation of desired compound (I)(-)-1,4-Dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 1-3)

(1) With use of7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester obtained in Example A-1 (4) and(-)-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxypyrrolidineobtained in Example B-3, there was obtained (+)-7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (amorphous) in the same manner as in Example C-1 (1).

a!_(D) ²⁹ +9.0° (c=1.002, chloroform)

(2) The above-captioned compound was obtained from the ethyl esterproduced in the above (1), according to the procedure described inExample C-1 (2).

Melting point: 278°-282° C. (decomposed)

a!_(D) ²⁹ -25.2° (c=0.504, water)

Examples C-4 and C-5 Preparation of desired compound (I)

The following compounds were obtained according to almost the sameprocedure as described in Example C-1.

    ______________________________________                                        Exam- Compound                                                                                 ##STR41##          Melting                                   ple   No.       Y'             Ra  X    point (°C.)                    ______________________________________                                        C-4                                                                                            ##STR42##     Et* --   253-259 (decom- posed)                      1-4       Same as above  H   HCl  263-269                                                                       (decom-                                                                       posed)                                C-5                                                                                            ##STR43##     Et  --   98-100                                      2         Same as above  H   HCl  268-271                                                                       (decom-                                                                       posed)                                ______________________________________                                         *Et denotes ethyl. (the same applies in the followings)                  

Example C-6 Preparation of desired product (I)7-(3-Amino-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 3) and its L-Ala derivative (Compound 3-1)

(1) Acetonitrile (10 ml) containing 3-aminopyrrolidine (1.6 g) was addeddropwise to a suspension composed of the ester (2.0 g) obtained inExample A-1 (4) and acetonitrile (80 ml). The resulting mixture wasstirred at room temperature for one hour and a half, and crystal wastaken by filtration. Thus,7-(3-amino-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (1.89 g) was produced by recrystallization from a mixedsolution composed of chloroform, methanol and diisopropylether.

Melting point: 219°-221° C.

(2) A suspension composed of the above ester (1.0 g) and a 10% aqueoussolution of hydrochloric acid (15 ml) was stirred at 95° C. for 3.5hours. Crystals were taken by filtration, and washed with a mixedsolution composed of chloroform and methanol to give7-(3-amino-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 3)(0.93 g).

Melting point: 267° C. (decomposed)

(3) To a mixture composed of the ethylester (2.1 g) obtained in theabove (1), N-t-butoxycarbonyl-L-alanine (1.5 g) and methylene chloride(80 ml), there was added 1.6 g of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC), andthe resulting mixture was stirred at room temperature for two hours.After washed with an aqueous solution of sodium hydrogen carbonate, themixture was dried over anhydrous sodium sulfate, and was thenconcentrated under reduced pressure, and was subsequently purified bysilica gel column chromatography (eluent chloroform:methanol=20:1) togive 7-3-(N-t-butoxycarbonyl-L-alanyl)amino-1-pyrrolidinyl!-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (2.9 g).

Melting point: 138°-140° C.

a!_(D) ²⁷ -46° (c=1.0, chloroform)

(4) A mixture composed of the ester (1.4 g) obtained in the above (3),0.5N hydrochloric acid (30 ml) and ethanol (16 ml) was stirred at 70° C.for two days. Solvent was concentrated under reduced pressure, and7-(3-L-alanylamino-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 3-1) (0.77 g) was obtained byrecrystallization from 10% hydrochloric acid and ethanol.

Melting point: 229°-231° C. (decomposed)

a!_(D) ²⁷ +10° (c=0.5, water)

Examples C-7-C-10 Preparation of desired product (I)

The following compounds were produced by almost the same method asmentioned in Example C-1.

    ______________________________________                                        Exam- Compound                                                                                 ##STR44##          Melting                                   ple   No.       Y'             Ra  X    point (°C.)                    ______________________________________                                        C-7                                                                                            ##STR45##     Et  --   95-97                                       4                                                                                        ##STR46##     H   HCl  297-299 (decom- posed)                C-8                                                                                            ##STR47##     Et  --   154-156                                     5                                                                                        ##STR48##     H   HCl  279-282 (decom- posed)                C-9                                                                                            ##STR49##     Et  --   157-159                                     6         Same as the above                                                                            H   HCl  266-270                                                                       (decom-                                                                       posed)                                C-10                                                                                           ##STR50##     Et  --   238-240                                     7                                                                                        ##STR51##     H   HCl  269-271 (decom- posed)                ______________________________________                                         *Boc denotes tbutoxycarbonyl. (the same applies in the followings)       

Example C-11 Preparation of desired product (I)7-(3-Amino-1-pyrrolidinyl)-1-(4-fluoro-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 8)

(1) A mixture composed of7-chloro-1-(4-fluoro-2-thiazoly)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid ethyl ester (30 mg) obtained in Example A-2,3-(t-butoxycarbonylamino)pyrrolidine (19 mg), triethylamine (26 mg) andacetonitrile (10 ml) was stirred at room temperature for 30 minutes.Solvent was distilled off under reduced pressure, and water was added,and then, the resulting mixture was extracted with chloroform. Theobtained extract was dried over anhydrous sodium sulfate, and solventwas distilled off under reduced pressure. The resulting residue wassubjected to recrystallization with use of ethyl acetate, and thus,there was obtained7-(3-t-butoxycarbonylamino-1-pyrrolidinyl)-1-(4-fluoro-2-thiazolyl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylicacid ethyl ester (40 mg).

Melting point: 233°-234° C.

(2) A solution composed of the above ester (40 mg) and 20% hydrochloricacid (2 ml) was heated to reflux for 1.5 hour. After cooling, crystalwas taken by filtration, and was then washed with dilute aqueoussolution of hydrochloric acid, and thus, the above-captioned Compound 8(32 mg) was obtained.

Melting point: 283°-284° C. (decomposed)

Example C-12 Preparation of desired product (I)5-Amino-7-(3-amino-1-pyrrolidinyl)-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 9)

(1)5-Amino-7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid (1.0 g) obtained in Example A-4 (2) and aminopyrrolidine (800 mg)were made to react in almost the same manner as in Example C-1, andthus, the above-captioned Compound 9 (610 mg) was produced.

Melting point: 261°-263° C.

Examples C-13 Preparation of desired product (I)

The following compounds were produced by almost the same method asmentioned in Example C-1.

    ______________________________________                                        Exam- Compound                                                                                 ##STR52##          Melting                                   ple   No.       Y'             Ra  X    point (°C.)                    ______________________________________                                        C-13                                                                                           ##STR53##     Et  --   248-251                                     10        Same as the above                                                                            H   HCl  243-246                               ______________________________________                                    

Example C-14 Preparation of desired product (I)3-Formyl-1,4-dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridinehydrochloride (Compound 11)

(1) Triethylamine (13.4 ml) was added to a suspension composed of7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (8.49 g) obtained in Example A-1 (4),trans-3-methoxy-4-methylaminopyrrolidine dihydrochloride (6.5 g) andacetonitrile (400 ml). After stirred overnight at room temperature, theresulting reaction mixture was concentrated under reduced pressure.Then, an aqueous solution of sodium hydrogen carbonate was added, andthe resultant mixture was extracted with chloroform. The obtainedextract was washed with saturated brine, and was then dried overanhydrous sodium sulfate, and thereafter, solvent was distilled offunder reduced pressure. Methylene chloride (500 ml) was added to theobtained residue, and, to the resultant mixture, di-t-butyl dicarbonate(6.4 g) was added under ice cooing. After stirred overnight at roomtemperature, the resulting reaction mixture was concentrated underreduced pressure, and the resultant residue was purified by silica gelcolumn chromatography (eluent chloroform:methanol=100:1) to give 7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester (9.1 g).

IR (KBr) cm⁻¹ : 1735, 1695

MS (m/z): 530 (MH⁺)

(2) A 1N aqueous solution of sodium hydroxide (150 ml) was added to amixture composed of the compound (8.95 g) obtained in the above (1) andethanol (150 ml), and the resulting mixture was stirred at the sametemperature for 30 minutes, and overnight at room temperature. Then, themixture was made acidic with an aqueous solution of acetic acid, and wasthen extracted with chloroform. The obtained extract was washed withsaturated brine, and was then dried over anhydrous sodium sulfate.Solvent was distilled off under reduced pressure, and thus, 7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid (7.19 g) was obtained.

Melting point: 185°-188° C.

IR (KBr) cm⁻¹ : 1715, 1690

MS (m/z): 502 (MH⁺)

(3) To a mixture composed of the compound (7.15 g) obtained in the above(2) and methanol (400 ml), there was added 2.16 g of sodium borohydrideunder ice cooling, and the resulting mixture was stirred for 15 minutesat the same temperature and then stirred overnight at room temperature.Solvent was distilled off under reduced pressure, and the obtainedresidue was extracted with chloroform. The resultant extract was washedwith saturated brine, and was then dried over anhydrous sodium sulfate.Solvent was distilled off under reduced pressure, and the obtainedresidue was subsequently purified by silica gel column chromatography(eluent chloroform:methanol=100:1) to give 7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-1,2,3,4-tetrahydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine(3.9 g).

IR (neat) cm⁻¹ : 1690

MS (m/z): 460 (MH⁺)

(4) The compound (3.8 g) obtained in the above (3) was dissolved i ntetrahydro furan (500 ml), and, to the resulting solution, there wasadded dropwise 6.1 ml of a solution of n-butylithium (1.6M) in n-hexaneat a temperature of -78° C. After the obtained solution was stirred for30 minutes at the same temperature, ethyl formate (1.34 ml) was added,and then, temperature was raised slowly, and the resultant mixture wasstirred overnight. Solvent was distilled off under reduced pressure, andan aqueous solution of acetic acid was added to the obtained residue,and then, the resulting mixture was extracted with chloroform. Theresultant extract was washed with saturated brine, and was then driedover anhydrous sodium sulfate. Solvent was distilled off under reducedpressure, and thus, 7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-3-formyl-1,2,3,4-tetrahydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine(3.87 g) was obtained.

IR (neat) cm⁻¹ : 1690, 1615

MS (m/z): 488 (MH⁺)

(5) The compound (3.6 g) obtained in the above (4) was dissolved in1,4-dioxane (160 ml), and, to the resulting solution,2,3-dichloro-5,6-dicyanobenzo-quinone (2.51 g) was gradually added.After the resultant mixture was stirred for 2.5 hours, solvent wasdistilled off under reduced pressure, and an aqueous solution of sodiumhydroxide was added to the obtained residue, and the resultant mixturewas extracted with chloroform. The resultant extract was washed withsaturated brine, and was then dried over anhydrous sodium sulfate.Solvent was distilled off under reduced pressure, and the obtainedresidue was subsequently purified by silica gel column chromatography(eluent chloroform:methanol=100:1) to give 7-trans-3-(N-t-butoxycarbonylmethylamino)-4-methoxy-1-pyrrolidinyl!-3-formyl-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine(1.73 g).

Melting point: 130°-132° C.

IR (KBr) cm⁻¹ : 1695, 1645, 1615

MS (m/z): 486 (MH⁺)

(6) A mixture composed of the compound (1.65 g) obtained in the above(5), a 10% aqueous solution of hydrochloric acid and ethanol (40 ml) washeated at 50°-60° C. for seven hours. Crystal was taken by filtration,and was then washed with ethanol and diisopropylether to give3-formyl-1,4-dihydro-7-(trans-3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridinehydrochloride (Compound 11) (1.05 g).

Melting point: 255°-263° C. (decomposed)

IR (KBr) cm⁻¹ : 3460, 1695, 1645

MS (m/z): 386 (MH⁺)

Examples C-5-C-27 Preparation of desired product (I)

The following compounds were produced according to the method mentionedin Example C-1.

    __________________________________________________________________________     Com- pound                                                                             ##STR54##              Melting point                                Example                                                                            No. A R.sub.s                                                                          R.sub.1                                                                         Y'         Ra                                                                              X  (°C.)                                  __________________________________________________________________________    C-15     CH                                                                              H  H                                                                                ##STR55## Et                                                                              -- 92-95                                              12  CH                                                                              H  H Same as the above                                                                        H HCl                                                                              245-248                                       C-16     CH                                                                              H  H                                                                                ##STR56## Et                                                                              -- amorphous                                          13  CH                                                                              H  H Same as the above                                                                        H HCl                                                                              293-295                                                                       (decomposed)                                  C-17     N H  H                                                                                ##STR57## Et                                                                              -- 228-230                                            14  N H  H Same as the above                                                                        H HCl                                                                              288-291                                                                       (decomposed)                                  C-18 15  CH                                                                              NH.sub.2                                                                         H                                                                                ##STR58## H -- 247-250                                       C-19     CH                                                                              H  F                                                                                ##STR59## Et                                                                              -- 198-199                                            16  CH                                                                              H  F Same as the above                                                                        H HCl                                                                              277-279                                                                       (decomposed)                                  __________________________________________________________________________     Com- pound                                                                              ##STR60##             Melting point                                Example                                                                             No. R.sub.1                                                                          R.sub.1 '                                                                       Y'         Ra                                                                              X   (°C.)                                  __________________________________________________________________________    C-20      H  H                                                                                ##STR61## Et                                                                              --  152-155                                             17  H  H                                                                                ##STR62## H HCl 296-299 (decomposed)                          C-21      F  H                                                                                ##STR63## Et                                                                              --  246-247                                             18  F  H Same as the above                                                                        H HCl 300 or higher                                 C-22      H  Cl                                                                               ##STR64## Et                                                                              --  228-229                                             19  H  Cl                                                                               ##STR65## H HCl 236-237                                       C-23      H  H                                                                                ##STR66## Et                                                                              --  236-238                                             20  H  H                                                                                ##STR67## H HCl 259-262 (decomposed)                          __________________________________________________________________________     Com- pound                                                                              ##STR68##             Melting point                                Example                                                                            No.  R.sub.3                                                                          Y'            Ra                                                                              X  (°C.)                                  __________________________________________________________________________    C-24      H                                                                                 ##STR69##    Et                                                                              -- 109-111                                            21   H                                                                                 ##STR70##    H HCl                                                                              300 or higher                                 C-25      CF.sub.3                                                                          ##STR71##    Et                                                                              -- 208-209                                            22   CF.sub.3                                                                         Same as the above                                                                           H HCl                                                                              291-292 (decomposed)                          C-26 23   Cl                                                                                ##STR72##    H HCl                                                                              300 or higher                                 C-27      H                                                                                 ##STR73##    Et                                                                              -- 128-132                                            24   H                                                                                 ##STR74##    H HCl                                                                              285-288 (decomposed)                          __________________________________________________________________________     *Ac: acetyl                                                              

Example C-28 Preparation of desired product (I)1,4-Dihydro-7-(trans-3-methylamino-4-methylthio-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid hydrochloride (Compound 25)

(1) With use of7-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester obtained in Example A-1 (4) andtrans-3-methylamino-4-methylthiopyrrolidine, there was obtained1,4-dihydro-7-(trans-3-methylamino-4-methylthio-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid ethyl ester in the same manner as in Example C-1 (1).

Melting point: 164°-165° C.

(2) The above-captioned compound was produced from the ethyl esterobtained in the above (1), in accordance with the process mentioned inExample C-1 (2).

Melting point: 271°-272° C.

4. Series D

Example D-1 Preparation of liquid agent

Prescription

    ______________________________________                                        Compound 1-1          2 g                                                     Sorbitol             50 g                                                     Sodium hydroxide     appropriate amount                                       Distilled water for injection                                                                      appropriate amount                                                            1000 ml                                                  ______________________________________                                    

Preparation method:

Compound 1-1 and sorbitol were dissolved in part of distilled water forinjection, and the residual distilled water was added so that pH of theresultant solution might be adjusted to be 4.0. This solution wasfiltered with a membrane filter (0.22 μm) to give a liquid forinjection.

Example D-2 Preparation of freeze-dried agent

Prescription

    ______________________________________                                        Compound 1-1         1 g                                                      Mannitol             5 g                                                      Sodium hydroxide     appropriate amount                                       Distilled water for injection                                                                      appropriate amount                                                            100 ml                                                   ______________________________________                                    

Preparation method

Compound 1-1 and mannitol were dissolved in part of distilled water forinjection, and the residual distilled water was added so that pH of theresultant solution might be adjusted to be 5.0. This solution wasfiltered with a membrane filter (0.22 μm), and the obtained filtrate wasfreeze-dried to give a powder agent for injection.

Industrial Applicability

The compound of this invention is useful as medicine, in particular asanti-tumor agent, for mammals including human beings.

We claim:
 1. A pyridone-carboxylic acid derivative having the followinggeneral formula (I), or a salt thereof: ##STR75## wherein R₁ is ahydrogen atom, a lower alkoxy group, a halogen atom, a lower alkyl groupwhich may be substituted with halogen atom, or a phenyl group which maybe substituted with halogen atom;R₂ is a carboxyl group or a groupconvertible to a carboxyl group; R₃ is a hydrogen atom, an amino groupwhich may be protected, a halogen atom or a lower alkyl group which maybe substituted with halogen atom; A is CH; m is an integer of 1 or 2;and Y is an eliminatable group selected from the group consisting of ahalogen atom, a lower alkoxy group, a lower alkylthio group, a loweralkylsulfinyl group, a lower alkylsulfonyl group, an arylsulfonyl group,a lower alkylsulfonyloxy group and an arylsulfonyloxy group, or a grouphaving the following formula Y' ##STR76## wherein R₄ is a hydrogen atomor a lower alkyl group; Z is a hydrogen atom, a lower alkyl group or a Zgroup convertible to a hydrogen atom;R₅ is a hydrogen atom, a halogenatom, a lower alkoxy group, a lower alkylthio group or a lower alkylgroup which may be substituted with halogen atom; n is an integer of 0or 1; and p is an integer of 1, 2, 3 or
 4. 2. A pyridone-carboxylic acidderivative or salt thereof of claim 1 wherein Y is an eliminatable groupin the above formula (I).
 3. A pyridone-carboxylic acid derivativehaving the following formula (I-a) or salts thereof: ##STR77## whereinR₁ is a hydrogen atom, a lower alkoxy group, a halogen atom, a loweralkyl group which may be substituted with halogen atom, or a phenylgroup which may be substituted with halogen atom;R₂ is a carboxyl groupor a group convertible to a carboxyl group; R₃ is a hydrogen atom, anamino group which may be protected, a halogen atom or a lower alkylgroup which may be substituted with halogen atom; R₄ is a hydrogen atomor a lower alkyl group; Z is a hydrogen atom, a lower alkyl group or agroup convertible to a hydrogen atom; R₅ is a hydrogen atom, a halogenatom, a lower alkoxy group, a lower alkylthio group or a lower alkylgroup which may be substituted with halogen atom;A is CH; m is aninteger of 1 or 2; n is an integer of 0 or 1; and p is an integer of 1,2, 3 or
 4. 4. A pyridone-carboxylic acid derivative of claim 3 whosesalts are either salts derived from the carboxyl group of R₂ of formula(I-a) or acid addition salts derived from the basic substituent groupportion which is bound to the 3-position of the 1-pyrrolidinyl group. 5.A pyridone-carboxylic acid derivative or salt thereof of claim 3 whereinthe group convertible to a carboxyl group in R₂ of formula (I-a) is aformyl group or an ester.
 6. A pyridone-carboxylic acid derivative orsalt thereof of claim 3 wherein the group convertible to a hydrogen atomin Z of formula (I-a) is an amino acid residue or a peptide residue. 7.A pyridone-carboxylic acid derivative having the following formula (I-b)or salt thereof: ##STR78## wherein R₁ is a hydrogen atom, a lower alkoxygroup, a halogen atom, a lower alkyl group which may be substituted withhalogen atom, or a phenyl group which may be substituted with halogenatom;R₃ ' is a hydrogen atom, amino group, halogen atom or lower alkylgroup which may be substituted with halogen atom; R₄ is a hydrogen atomor a lower alkyl group; Z' is a hydrogen atom or a lower alkyl group; R₅is a hydrogen atom, a halogen atom, a lower alkoxy group, a loweralkylthio group or a lower alkyl group which may be substituted withhalogen atom; A is CH; m is an integer of 1 or 2; n is an integer of 0or 1; and p is an integer of 1, 2, 3 or
 4. 8. A pyridone-carboxylic acidderivative or salt thereof of claim 7 wherein both m and p are 1, and nis 0 in formula (I-b).
 9. A pyridone-carboxylic acid derivative or saltthereof of claim 7 wherein A is CH, both m and p are 1, n is 0, R₁ is ahydrogen atom or a fluorine atom, R₃ ' is a hydrogen atom, R₄ is ahydrogen atom or a lower alkyl group, Z' is a hydrogen atom, R₅ is alower alkyl group or a lower alkoxy group in formula (I-b).
 10. Apyridone-carboxylic acid derivative of claim 1 or 3 which is1,4-dihydro-7-(3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid or a salt thereof.
 11. A pyridone-carboxylic acid derivative ofclaim 10 which is either cis isomer or trans isomer of1,4-dihydro-7-(3-methoxy-4-methylamino-1-pyrrolidinyl)-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid, or an optically active substance thereof, or a salt thereof. 12.An anti-tumor agent which contains, as an effective ingredient, either apyridone-carboxylic acid derivative of formula (I-a) of claim 3 aphysiologically acceptable salt thereof.
 13. An anti-tumor agent whichcontains, as an effective ingredient, either the pyridone-carboxylicacid derivative of formula (I-b) of claim 7 or a physiologicallyacceptable salt thereof.
 14. The anti-tumor agent of claim 12 or 13which is an agent for the treatment or prophylaxis of non-solid tumor orsolid tumor.
 15. The anti-tumor agent of claim 14 wherein said non-solidtumor is leukemia or malignant lymphoma.
 16. The anti-tumor agent ofclaim 14 wherein said solid tumor is one which occurs in tissues oflung, breast, stomach, skin, ovary, uterus, intestine, urinary bladder,nasopharynx, head and neck, esophagus, liver, biliary tract, pancreas,kidney, testis, prostate, bone or brain.
 17. A pharmacologicalcomposition which comprises a pyridone-carboxylic acid derivative offormula (I-a) of claim 3 or of formula (I-b) of claim 7 orphysiologically acceptable salt thereof, and a pharmacologicallyacceptable carrier.
 18. The composition of claim 17 wherein said carrieris a solvent.
 19. The pharmacological composition of claim 17 which isin the form of a solution.
 20. The pharmacological composition of claim19 which is an agent for injection or transfusion.
 21. Thepharmacological composition of claim 17 which is a freeze-driedpreparation.